Journal of Bioscience and Bioengineering

VOL. 109 No. 3, 235 – 239, 2010

www.elsevier.com/locate/jbiosc

Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica

(Bacillariophyceae): Effect of some environmental factors

Stephen L. Pahl,1 David M. Lewis,1,⁎ Feng Chen,2 and Keith D. King1

School of Chemical Engineering, The University of Adelaide, Adelaide, South Australia 5005, Australia 1 and School of Biological Sciences, The University of
Hong Kong, Pokfulam Road, Hong Kong, P. R. China 2

Received 25 May 2009; accepted 13 August 2009

Available online 17 September 2009

To investigate the nutritional value of the diatom Cyclotella cryptica (Reimann, Lewin, and Guillard) as an alternative feed
for use in the aquaculture industry, the heterotrophic growth characteristics and resultant fatty acid profile of the microalga
were studied when cultivated under a variety of controlled salinity and temperature conditions. In addition, the effects of pH
on the growth characteristics were also studied. The maximum specific growth rate was affected by initial pH and cultivation
temperature, but not by salinity. The optimal pH and temperature ranges for growth were 7.2 to 8.1 and 22.5 to 25.0 °C,
respectively. Lipid accumulation and the fatty acid composition were also affected by cultivation temperature and salinity. The
optimal temperature range and salinity level for lipid accumulation were 18.0 to 25.0 °C and 11.2 psu, respectively. In all cases
the fatty acid distribution was similar, with the most abundant fatty acids being palmitic acid (16:0), palmitoleic acid (16:1
n-7), stearidonic acid (18:4 n-3, SDA), eicosapentaenoic acid (20:5 n-3, EPA), and decosahexaenoic acid (22:6 n-3, DHA).
© 2009, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Cyclotella cryptica; Heterotrophic; Salinity; Temperature; pH; PUFAs]

The production of live microalgae is a major ‘bottleneck’ in the
aquaculture industry and has prompted the search for superior
microalgal strains, improved cultivation conditions, improved culture
systems, and the development of alternative feed sources. Despite
recent advances into alternative feed sources, microalgae are the
biological starting point for energy flow through aquatic ecosystems (1)
and remain an essential feed source for commercially farmed mollusks,
crustaceans, and fish species, for at least part of their life cycle.
The nutritional value of microalgal biomass, as feed for aquatic
organisms, depends on several attributes including microalgae size
and shape, digestibility, non-toxicity, and biochemical composition
and should match the nutritional requirements of the target
organism (2, 3). The growth rate and resulting biochemical
composition of microalgae is influenced by environmental conditions
(i.e. light, temperature, salinity, pH, and aeration), nutritional factors
(source and availability), and culture age (2, 4, 5). In addition,
economic and practical constraints require reliable microalgae
cultivation systems producing consistent and high-quality microalgal
biomass at low cost.
Traditional techniques, which rely on photosynthetic microalgae
grown in outdoor ponds or indoors under artificial lights, suffer from the
phenomenon of light-limitation and are often unreliable as they are
frequently subject to inexplicable crashes. Microalgae production cost
Abbreviations: DHA, docosahexaenoic acid (22:6 n-3); EPA, eicosapentaenoic acid
(20:5 n-3); PUFAs, polyunsaturated fatty acids.
⁎ Corresponding author. Tel.: +61 8 8303 5503; fax: +61 8 8303 4373.
E-mail address: david.lewis@adelaide.edu.au (D.M. Lewis).
can be as high as US$ 600 kg−1 and several hatcheries have estimated
that, on average, 30%–40% of their total operational costs may be directly
attributed to the cultivation of microalgal biomass (6). The excessive
production costs are due to the relatively low biomass concentrations
achieved, large operating costs, and culture inefficiencies.
A number of recent reports including Brown (7), Duerr et al. (8),
Harel et al. (9), and Heasman (10) have outlined the potential of
heterotrophically grown microalgal biomass for aquaculture feeds.
Heterotrophic production may provide a cost-effective alternative for
the cultivation of microalgae species, which are capable of utilizing
organic carbon substrates as their sole carbon and energy source. This
mode of growth has several advantages as it eliminates the
requirement for light and therefore the phenomenon of light-
limitation and offers the possibility of greatly increasing the biomass
concentration, thus reducing the volume required to achieve a given
production level. In addition, heterotrophic microalgae can be grown
on a large scale using well-developed technology in conventional
fermentors. However, heterotrophic cultivation has its disadvantages,
including the following: 1) there are a limited number of microalgae
species can have the ability to utilize organic compounds, 2) potential
contamination by bacteria, 3) growth inhibition at low organic
substrate concentrations, and 4) heterotrophic cultivation can modify
the chemical composition (11). While challenges exist, the high
degree of process control inherent in typical fermentors may result in
improved biomass productivity with a more consistent and repro-
ducible biochemical quality product. Commercial-scale cultivation of
heterotrophic microalgae exists in Japan, Taiwan, and the United
States (12); however, only a limited number of heterotrophic strains,
including Crypthecodinium sp. and Schizochytrium sp. (Aquafauna Bio-

1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2009.08.480
236 PAHL ET AL. J. BIOSCI. BIOENG.,

FIG. 1. Heterotrophic growth curves of C. cryptica at six salinity levels (data are
expressed as mean ± standard deviation, n = 2); temperature 25.0 °C; pH 7.5.
Marine, Inc. CA, USA and Advanced BioNutrition Corp., MD, USA) are
currently grown commercially for use as aquaculture feeds.
The diatom Cyclotella cryptica is capable of heterotrophic growth
(13–15), has previously been trialed as part of an artificial diet in the
development of juvenile mollusks (16), and has been recommended as
a species worthy of further investigation. Environmental conditions
have a strong influence on cell metabolism (growth, maintenance, and
biochemical composition) and many responses are species-specific
and should be individually studied. The aim of the present work was to
determine the heterotrophic growth characteristics and fatty acid
composition of C. cryptica when cultivated under a variety of
controlled salinity and temperature conditions and to determine the
growth rate when C. cryptica was cultivated at different pH conditions.
MATERIALS AND METHODS
Microalgae and culture media The diatom C. cryptica (UTEX 1269) was
cultured in a modification of the SK medium reported by Gladue and Maxey (13). The
base media consisted of (per liter) 6.8 g synthetic sea salt (Taikong Corp., Taipei,
Taiwan), 2.17 g MgSO4·7H2O, 1.6 g tryptone, 917 mg NaNO3, 800 mg yeast extract,
50.5 mg KH2PO4, 34 mg H3BO3, 20 mg FeSO4·7H2O, 15 mg NaH2PO4·2H2O, 6 mg
thiamine·HCl, 5 mg Na2EDTA, 4.3 mg MnCl2·4H2O, 0.3 mg vitamin B12, 0.3 mg biotin,
0.3 mg ZnCl2, 0.26 mg NiSO4·6H2O, 0.13 mg CoCl2·6H2O, 0.03 mg Na2MoO4·2H2O,
0.017 mg Na2SeO3, and 0.01 mg CuSO4·5H2O and was supplemented with 10 g·L−1
glucose and 480 mg·L−1 Na2SiO3·5H2O. The pH of the medium was adjusted to 7.5
prior to autoclaving at 121 °C for 20 min.
Experiments were performed to test the effects of salinity and temperature on the
growth dynamics and fatty acid composition of this species. The salinity was tested at six
levels (3.4, 6.8, 11.2, 17.0, 27.2, and 34.0 psu) by modifying the quantity of synthetic sea
salt used. The cultivation temperature was controlled at six temperatures (12.5, 15.0, 18.0,
22.5, 25.0, and 33.0 °C) by adjusting the thermostat on each incubator. A subsequent
experiment was also undertaken to examine the effect of initial pH on the heterotrophic
growth rate. The pH of the culture media was tested at seven different values (7.2, 7.5, 7.8,
8.1, 8.4, 8.7, and 9.0) by the addition of varying ratios of Trizma Base (Sigma Inc., St. Louis,
MO, USA) and Trizma HCl (Sigma Inc., St. Louis, MO, USA). Trizma Base and Trizma HCl
solutions were filter sterilized and added to the culture media after it was autoclaved.
Contamination was prevented by maintaining aseptic techniques. In all cases the Tris (2-
amino-2-hydrxymethyl-1,3-propanediol) concentration was 0.05 M. All cultures were
grown in 250 mL Erlenmeyer flasks containing 100 mL sterilized medium (6.8 psu; initial
pH 7.5) and were inoculated with 5%–10% vol./vol. exponentially growing culture and
were incubated in the dark at 25 °C on an orbital shaker (100 rpm), unless otherwise
indicated. Variable inoculum volumes were used to ensure that the biomass concentration
at the commencement of each investigation was standardized. Cultures were harvested
during the exponential phase, unless otherwise indicated.
Determination of biomass and growth rate The biomass concentration was
determined spectrophotometrically from the culture absorbance measured at 675 nm
and was compared to a correlation curve that was previously developed (Pahl,
unpublished data). At the termination of the experiment, the biomass was harvested by
centrifugation (15,700 rcf [relative centrifugal force] for 10 min), rinsed twice with
reverse osmosis water, dried overnight in a SpeedVac Concentrator (Savant Instru-
ments Inc., Farmingdale, NY, USA), and stored at − 18 °C prior to analysis. The growth
rate was determined by plotting the natural logarithm of the biomass concentration
against time. Readings within the exponential phase were then used to obtain the
maximum specific growth rate by linear regression.
Lipid analysis Fatty acid methyl esters (FAMEs) were prepared by direct
transesterification as reported by Christie (17). Lipids were dissolved in toluene and
subjected to methanolysis (1% vol./vol. of H2SO4 in methanol) overnight at 50 °C. After
cooling, the resulting FAMEs were extracted with n-hexane and washed with
potassium bicarbonate (2% wt./vol.) prior to being evaporated to dryness under a
stream of nitrogen. The FAMEs were re-dissolved in n-heptane and further dried over
anhydrous sodium sulfate. The FAMEs were separated using a Hewlett-Packard 6890
gas chromatograph equipped with a flame ionization detector and a BPX-70 (50 m
× 0.32 mm ID × 0.25 μm film thickness) capillary column (SGE Pty Ltd, Melbourne,
Victoria, Australia). Helium was used as the carrier gas with a column flow rate of
2.4 mL/min. The injector was kept at 250 °C, with an injection volume of 5 μL and the
split ratio was set at 20:1. The initial column temperature was 140 °C and programmed
to rise to 220 °C at 5 °C/min, held for 1 min, followed by a further rise to 260 °C at 20 °C/
min. The detector temperature was kept at 300 °C. FAMEs were identified by
comparison of known retention times and were quantified based on the external
reference standard GLC-463 (Nu-Chek Prep, Elysian, MN, USA). Total lipid content was
calculated by summation of individual fatty acids.
Statistical analysis Significant differences between experimental conditions
were detected by means of one-way analysis of variance (ANOVA), followed by
pairwise comparisons using Tukey's test, where appropriate. Equality of variance was
checked using the modified Levene procedure.
RESULTS
C. cryptica grew heterotrophically under all of the conditions
investigated and the cells remained dark brown in color. No

TABLE 1. Growth rate and fatty acid composition of C. cryptica grown heterotrophically at six salinity levels (data are expressed as mean ± standard deviation, n = 2); temperature
25.0 °C; pH 7.5.
Salinity level (psu)

3.4 6.8 11.2 17.0 27.2 34.0

−1
Growth rate (hr ) 0.030 ± 0.000 0.031 ± 0.001 0.030 ± 0.000 0.031 ± 0.004 0.038 ± 0.002 0.029 ± 0.004
Fatty acids (mg·g−1) a
14:0 2.3 ± 0.1 2.1 ± 0.1 2.6 ± 0.4 1.7 ± 0.1 1.4 ± 0.1 1.3 ± 0.1
15:0 tr b tr 1.1 ± 0.1 1.1 ± 0.1 tr tr
Unknown 1 1.5 ± 0.1 1.4 ± 0.0 1.6 ± 0.2 1.2 ± 0.1 1.2 ± 0.1 1.1 ± 0.0
16:0 18.4 ± 1.9 16.0 ± 0.4 20.7 ± 3.0 17.7 ± 1.0 15.2 ± 2.5 12.4 ± 0.4
16:1 (n-7) 15.3 ± 1.3 11.1 ± 1.8 19.6 ± 4.9 10.6 ± 0.8 10.7 ± 0.3 11.7 ± 0.2
18:1 (n-9) 1.2 ± 0.5 1.4 ± 0.6 1.4 ± 0.0 3.9 ± 0.3 3.9 ± 0.9 1.1 ± 0.4
18:3 (n-6) 1.8 ± 0.1 1.7 ± 0.0 1.4 ± 0.0 1.2 ± 0.1 1.2 ± 0.1 tr
18:4 (n-3) 3.9 ± 0.5 3.9 ± 0.1 4.1 ± 0.2 3.5 ± 0.4 3.5 ± 0.3 2.3 ± 0.0
20:5 (n-3) 10.9 ± 0.5 11.3 ± 1.1 13.5 ± 1.2 9.7 ± 0.8 9.7 ± 1.1 10.9 ± 0.2
22:6 (n-3) 1.4 ± 0.1 1.8 ± 0.0 1.7 ± 0.1 1.7 ± 0.2 1.7 ± 0.2 1.3 ± 0.0
Total lipids (mg·g−1) 62.9 ± 5.3 57.0 ± 2.7 74.0 ± 10.6 58.4 ± 3.8 58.4 ± 6.5 48.0 ± 0.4
Unsat c 60.5 ± 0.6 61.1 ± 1.0 61.6 ± 0.2 58.2 ± 0.9 59.7 ± 1.0 64.4 ± 0.8
Δ / mole d 1.7 ± 0.0 1.9 ± 0.0 1.7 ± 0.1 1.7 ± 0.0 1.7 ± 0.0 1.9 ± 0.0
a
Fatty acids are denoted by C:X (n-y) notation, where C is the number of carbon atoms, X is the number of double bonds, and y is the position of the first double bond counted from
the methyl terminal. Fatty acids less than 1 mg·g−1 dry cell weight were excluded.
b
tr: indicates that the fatty acid was present, but at a concentration below 1 mg·g−1 dry cell weight.
c
Unsat: unsaturated fatty acids (percent of total lipids).
d
Δ/mole: degree of fatty acid unsaturation = (1.0(% monoenes) + 2.0(% dienes) + 3.0(% trienes) + 4.0(% tetraenes) + 5.0(% pentaenes) + 6.0(% hexaenes)) / 100.
VOL. 109, 2010
FIG. 2. Gas chromatogram of the fatty acid methyl esters from the lipids extracted of heterotrophically grown C. cryptica; salinity 11.2 psu; temperature 25.0 °C; initial pH 7.5. Peaks
greater than 1 mg·g−1 are labeled using the C:X (n-y) notation, where C is the number of carbon atoms, X is the number of double bonds, and y is the position of the first double bond
counted from the methyl terminal.
contamination was visualized in any of the cultures. The heterotro-
phic growth curves when C. cryptica was cultivated at six salinity
levels are shown in Fig. 1. The maximum specific growth rates and
fatty acid compositions of C. cryptica grown at the different salinity
levels are reported in Table 1. The maximum specific growth rate was
not significantly affected (P N 0.05) by the salinity of the media within
the range of 3.4 to 34.0 psu. However, a lag phase of 2 days was
observed (see Fig. 1) when the inoculum, which was cultured at
27.2 psu, was transferred into fresh media at a salinity level of 3.4 psu.
There were significant variations (P N 0.05) in lipid content, degree of
unsaturation, and EPA synthesis between various salinity treatments.
The maximum lipid and EPA content occurred when the salinity was
11.2 psu. The chromatogram of the fatty acid extract, shown in Fig. 2,
highlights the fatty acids that were synthesized in concentrations
above 1 mg·g−1 and includes one peak, which was unidentifiable. In
all salinity treatments the principal fatty acids were palmitic acid
(16:0), palmitoleic acid (16:1 n-7), stearidonic acid (18:4 n-3, SDA),
eicosapentaenoic acid (20:5 n-3, EPA), and decosahexaenoic acid
(22:6 n-3, DHA).
The heterotrophic growth curves when C. cryptica was cultivated
at seven initial pH values are shown in Fig. 3. The maximum specific
growth rates and pH changes are shown in Fig. 4. The maximum
specific growth rate of C. cryptica was affected by the initial pH of the
culture media. While the study was preliminary, the optimal pH for
growth of C. cryptica was between 7.2 and 8.1.
The heterotrophic growth curves when C. cryptica was cultivated
at six temperatures are shown in Fig. 5. The maximum specific growth
rate and fatty acid composition of C. cryptica cultivated at the different
FIG. 3. Heterotrophic growth curves of C. cryptica at seven initial pH values;
temperature 25.0 °C; pH 7.5.
ENVIRONMENTAL FACTORS ON HETEROTROPHIC CYCLOTELLA 237
temperatures are reported in Table 2. The maximum specific growth
rates were significantly affected (P N 0.05) by cultivation temperature.
Extreme high and low temperatures resulted in thermal stress. The
temperature for fastest growth ranges from 22.5 to 25.0 °C. The
maximum specific growth rate declined if the temperature was
maintained above or below this range. Limited biomass at the
termination of the investigation meant that the fatty acid composition
from biomass cultivated at 12.5 °C could not be accurately determined
and is not reported in Table 2. Due to an equipment malfunction the
biomass cultivated at 22.5 °C had stopped growing between days 3
and 4 (see Fig. 5) and consequently the fatty acid composition for
22.5 °C is excluded from Table 2. There was no significant difference in
the overall lipid content when the cultivation temperature was held at
18.0, 25.0, and 33.0 °C (P N 0.05). The fatty acid profile remained
relatively consistent when the temperature was held at 15.0, 18.0, and
25.0 °C. However, the principle fatty acids in each treatment were
palmitic acid (16:0), palmitoleic acid (16:1 n-7), stearidonic acid
(18:4 n-3, SDA), eicosapentaenoic acid (20:5 n-3, EPA), and
decosahexaenoic acid (22:6 n-3, DHA).
DISCUSSION
Salinity Culture salinity interacts with nutrient dynamics
(nutrient availability, requirements, and uptake rates) in saline
systems and is known to affect microalgal productivity and compo-
sition (18). The current research showed that under the heterotrophic
FIG. 4. Maximum specific growth rate and pH changes when C. cryptica was
heterotrophically grown at seven initial pH values; temperature 25.0 °C; salinity
6.8 psu.
238 PAHL ET AL. J. BIOSCI. BIOENG.,

region reported by Hellebust (21). Hellebust (21) investigated the

FIG. 5. Heterotrophic growth curves of C. cryptica at six temperatures (data are
expressed as mean ± standard deviation, n = 2); salinity 6.8 psu; pH 7.5.
condition investigated C. cryptica is euryhaline. This agrees with
previous research where Liu and Hellebust (19) reported that under
photoautotrophic growth C. cryptica also grew well over a wide range
of salinities. The total lipid content and degree of fatty acid
unsaturation was significantly affected (P N 0.05) by the salinity. The
maximum lipid content occurred when the salinity was 11.2 psu.
While C. cryptica can grow over a range of salinity levels, the cells are
sensitive to rapid salinity changes. The lag phase experienced at the
lowest salinity was most likely due to acclimatization of the cells to
the new environmental conditions.
pH To determine the effect of pH on the growth characteristics
of C. cryptica, the algae was cultivated at various pH with the addition
of 0.05 M Tris. Tris at 0.05 M was used as the buffering agent as it is a
common buffer in marine media preparation (20). Previous experi-
ence had shown that 0.05 M Tris does not inhibit or supplement the
growth of C. cryptica under heterotrophic conditions. The experiment
was centered about pH 8, as in nature diatoms are highly abundant in
oceanic environments, which typically have a pH value close to 8.
While C. cryptica grew over an initial pH range from 7.2 to 9.0, the
optimal pH range was 7.2 to 8.1. This optimal pH range lies within the
glucose uptake rates for C. cryptica and reported that the glucose
uptake rates were highest when the pH was between 7 and 9. The
reduction in the maximum specific growth rate at a sub-optimal pH
value is likely a result of C. cryptica attempting to maintain its own
intracellular pH value. Intracellular pH is regulated by energy
dependent systems (22) and the cultivation at a sub-optimal pH
value would require a greater proportion of energy being diverted
into maintenance systems, and thus less energy is available for
biomass production. The investigation of initial pH was preliminary in
nature and was designed to determine the sensitivity of C. cryptica to
different initial pH values. pH sensitivity is an important parameter in
photoautotrophic and heterotrophic cultivation conditions.
Temperature Temperature is an important environmental
factor, which strongly influences microalgal growth, productivity,
and nutritional value. Marine microalgae generally respond to a
decrease in temperature by accumulating lipids and increasing the
ratio of unsaturated to saturated fatty acids. While a reduction in
cultivation temperature did not have a significant effect (P N 0.05) on
overall lipid accumulation in C. cryptica, the percent unsaturation and
degree of fatty acid unsaturation increased at lower temperatures,
provided that the cells were not thermally stressed. The increased
availability of molecular oxygen at the lower cultivation temperatures
is likely to be responsible for the increased fatty acid unsaturation
because oxygen dependent enzymes are involved in the desaturation
and elongation of lipids (23, 24). The optimal temperature for growth
was between 22.5 and 25.0 °C. While this temperature range is quite
narrow, it is not uncommon. Payer et al. (25) investigated the
temperature effects on 34 microalgal species and reported that while
some species have a wide optimal temperature range; other species
have a narrow optimal temperature range. The fact that C. cryptica
remained viable and grew over a wide temperature range (15.0 to
33.0 °C) is promising for commercial aquaculture applications.
Growth rates, final biomass, and fatty acid composition While
the heterotrophic growth rates reported in this study were slower
than the maximum reported photoautotrophic growth rate, with a
doubling time of approximately 6.5 hr (26), they were comparable to

TABLE 2. Growth rate and fatty acid composition of C. cryptica grown heterotrophically at six temperatures (data are expressed as mean ± standard deviation, n = 3, except where
stated); pH 7.5; salinity 6.8 psu.
Temperature (°C)

12.5 15.0 18.0 22.5 25.0 33.0

Growth rate (hr−1) 0.005 ± 0.001 0.019 ± 0.000 0.026 ± 0.001 0.033 ± 0.000 0.032 ± 0.001 0.023 ± 0.001
Fatty acids (mg·g−1) a
14:0 tr b 1.4 ± 0.3 2.1 ± 0.1 1.8 ± 0.2
15:0 tr tr tr 8.6 ± 2.2
Unknown 1 1.1 ± 0.1 1.3 ± 0.2 1.0 ± 0.0 tr
16:0 10.3 ± 0.4 16.1 ± 2.5 17.0 ± 0.6 12.9 ± 2.1
17:0 tr tr tr 1.3 ± 0.3
16:1 (n-9) tr tr tr 1.3 ± 0.3
16:1 (n-7) 8.4 ± 0.4 8.1 ± 1.3 6.4 ± 0.3 6.5 ± 1.5
Unknown 2 nd c nd tr 2.3 ± 0.6
18:1 (n-9) tr 1.9 ± 0.3 2.8 ± 0.3 4.8 ± 0.7
18:2 (n-9) tr tr tr 1.1 ± 0.2
18:3 (n-6) tr 1.5 ± 0.3 1.3 ± 0.1 tr
18:4 (n-3) 3.0 ± 0.2 4.6 ± 0.9 4.5 ± 0.3 tr
20:5 (n-3) 9.1 ± 0.8 11.1 ± 2.2 10.5 ± 0.5 3.9 ± 0.7
22:6 (n-3) 1.7 ± 0.1 2.2 ± 0.4 2.5 ± 0.1 tr
Total lipids (mg·g−1) 40.2 ± 2.6 54.9 ± 9.2 56.6 ± 1.5 50.2 ± 8.9
Unsat d 64.4 ± 0.4 60.4 ± 1.5 57.9 ± 0.9 42.0 ± 1.4
Δ / mole e 2.1 ± 0.0 2.0 ± 0.1 1.9 ± 0.1 0.9 ± 0.1
a
Fatty acids are denoted by C:X(n-y) notation, where C is the number of carbon atoms, X is the number of double bonds, and y is the position of the first double bond counted from
the methyl terminal. Fatty acids less than 1 mg·g−1 dry cell weight were excluded.
b
tr: indicates that the fatty acid was present, but at a concentration below 1 mg·g−1 dry cell weight.
c
nd: not detected.
d
Unsat: unsaturated fatty acids (percent of total lipids).
e
Δ / mole: degree of fatty acid unsaturation = (1.0(% monoenes) + 2.0(% dienes) + 3.0(% trienes) + 4.0(% tetraenes) + 5.0(% pentaenes) + 6.0(% hexaenes)) / 100.
VOL. 109, 2010
literature values for heterotrophic cultivation (13–15, 27). The
biomass was harvested during the exponential phase as continuous
cultivations are frequently used in aquaculture facilities. Continuous
cultivations are beneficial for aquaculture operations as biomass is
generally required on a continual basis and continuous harvesting
may minimizing any rapid changes in the biochemical composition
and nutritional value associated with the onset of the stationary
phase. Consequently, this study did not seek to determine the
maximum biomass concentration achievable under heterotrophic
cultivation conditions. The maximum biomass concentration recorded
in this study was 1.5 g·L−1, which is higher than the 0.5 to 1.0 g·L−1
typically observed in many aquaculture facilities. Previous experience
has indicated that sodium metasilicate was the most limiting nutrient
and continuing the cultivations would have resulted in the
development of silicate-limited growth conditions. While total lipid
content may rise during the stationary growth phase, especially when
silicate-limited (28, 29), increases in the total lipid content are often
a result of the accumulation of neutral lipids, which generally have a
lower degree of unsaturation and a lower concentration of essential
fatty acids. The major fatty acids synthesized by the diatom C.
cryptica under the heterotrophic conditions investigated were
palmitic acid (16:0), palmitoleic acid (16:1 n-7), stearidonic acid
(18:4 n-3, SDA), eicosapentaenoic acid (20:5 n-3, EPA), and
decosahexaenoic acid (22:6 n-3, DHA). These fatty acids are similar
to earlier reports where the cells were grown photoautotrophically
(28, 30) with the exception of hiragonic acid (16:3 n-3) which was
not identified. While hiragonic acid is not regarded as an essential
fatty acid, its non-detection is interesting as it may indicate a change
in the fatty acid elongation and desaturation pathways. This,
however, requires further investigation.
The consistent supply of microalgae remains a major bottleneck
for many commercial hatchery and nursery aquaculture operations
and more productive microalgae cultivation techniques are desirable.
The heterotrophic cultivation of microalgae has several advantages
and the use of heterotrophic biomass has significant potential as an
alternative feed in some aquaculture operations.
It has been well documented that the proximate biochemical
composition and fatty acid profile of microalgal biomass are
dependent on the microalgal strain, growth phase, environmental
conditions, and nutrient factors. The effects of cultivation tempera-
ture, culture salinity, and culture pH on the growth of C. cryptica were
examined and the resulting fatty acid compositions were identified.
The major fatty acids synthesized by C. cryptica under the heterotro-
phic growth (current study) where similar to the major fatty acids
synthesized under photoautotrophic conditions and included EPA and
DHA. While information on fatty acid profiles of a feed source can aid
in determining its potential suitability, ultimately the potential of a
feed source can only be gauged from feeding trials. These fatty acids
are important in the aquaculture industry and consequently hetero-
trophically cultivated C. cryptica could potentially be used as an
alternative feed source within some aquaculture industries.
ACKNOWLEDGMENTS
The authors wish to thank the Child Nutrition Research Centre
(Flinders Medical Centre, Australia) for the fatty acid analyses and the
School of Molecular and Biomedical Sciences (The University of
Adelaide, Australia) and Mr Mark Mano (CSIRO Molecular and Health
Technologies, Australia) for allowing access to equipment.
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