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International Journal of Marine Science 2013, Vol.3, No.17, 135-144
http://ijms.sophiapublisher.com
138
was also noted. A 15µL aliquot of 7.8 µM Nile Red
dissolved in acetone was added. A second fluorescence
of the stained samples were measured for 600 seconds
or until a peak fluorescence reading was indicated.
2.6.2 FTIR spectroscopy
For FTIR spectroscopy a 50 mL sample was taken
from each replicate flask for each treatment in the end
of cell cultivation, mixed and centrifuged at 3000 rpm
for 10 mins. The supernatant removed and the cells
re-suspended in approximately 100 µL of distilled
water, 30 µL of which was then deposited on CFR
Low-e microscope slide. The samples were then
desiccated under vacuum for overnight. The dried
cellular deposit on the CFR Low-e microscope slide
was placed on the FTIR microscope stage for spectral
acquisition (Chen et al., 2010; Giordano et al., 2001;
Wagner et al., 2010). FTIR spectra collected using
FTIR spectrometer (Varian 7000, FTIR Stingray
Imaging Series) coupled to an infrared microscope
(Varian 6000 UMA FTIR microscope) and equipped
with a mercury-cadmium-telluride detector cooled
with liquid N
2
. Spectra were collected over the
wavenumber range 4000 – 700 cm
-1
. The Varian FTIR
system was controlled by a Window-compatible PC
running Spectra Pro software.
2.6.3 Gravimetric determination of neutral lipids
To determine the total content of lipids in cells, 50 mL
of cells were harvested by centrifugation (4000 g for
20 min, Heraues Multifuge 3SR) in the end of cell
cultivation. Lipid were extracted by adding 28 mL of
50 mM phosphate buffer (pH 7.4) into the cell pellets,
resuspended and sonicated for 1 min. The samples
were transferred to a separatory funnel, added 35 mL
chloroform and 70 mL methanol and shaked rigorously
and leaved for 18 hours. After 18 hours has passed,
the samples were added by 35 mL chloroform and 35
mL distilled water, respectively. Allowed the sample
to stand for a further 18 hours and collect the lower
chloroform layer in a round bottom flask which
having previously recorded the weight of the empty
flask. The extract was evaporated in a water bath
(50
℃
) using a rotary evaporator (Buchi, Switzerland)
to remove solvents and weight the flask for determining
lipid content. The lipid extraction followed the
protocol reported by Guckert et al (1988); Wagner et
al (2010) and Lee et al (1998).
2.7 Statistical analysis
Mean comparison were conducted by one-way
analysis of variance (ANOVA), followed by LSD
test to determine significance. In all cases, comparisons
that showed a p value <0.05 were considered
significant.
3 Results and Discussion
3.1 Impact of nutrient depletion and temperature
stress on algae biomass
The impact of nutrient depletion and temperature
stress was examined on three biomass indicators, cell
density, relative growth rate, chlorophyll
a
concentration
and dry weight.
Dunaliella tertiolecta, Nannochloropsis
sp. and
Scenedesmus
sp. were grown in batch culture
under three nutrient conditions: nutrient replete, no
nitrate and no phosphate and two temperature
conditions: 18
℃
and 25
℃
.
Figure 1, 2 and 3 illustrates the effect of nutrient and
temperature cultivation conditions on the (a) cell
growth and (b) Chl
a
content of three microalgae. The
growth behavior of
Dunaliella tertiolecta, Scenedesmus
sp. and
Nannochloropsis
sp. under different nutrient
and temperature conditions showed a varied trend
(Figure 1). Cell growth in terms of cell density was
higher at control treatment and normal temperature
than under nutrient depletion and temperature stress.
The peak of cell density all microalgals cultured were
occurred at day 6 or day 7. The effect of temperature
on cell density of microalgae varied within species.
Cell density was higher at ambient temperature for
marine (
Dunaliella tertiolecta
and
Nannochloropsis
sp.) and freshwater species (
Scenedesmus
sp.), such as
18
℃
and 25
℃
, respectively.
Dunaliella tertiolecta
showed a higher cell density at 18
℃
than 25
℃
.
Eighteen degree of temperature for
Dunaliella
tertiolecta
showed an optimum temperature for cell
growth.
Dunaliella tertiolecta
has a lower temperature
for optimum growth than others species (
Dunaliella
salina
and
D. viridis
), such as 22
℃
and 26
℃
,
respectively (Garcia et al., 2007). Like others species
of Dunaliella,
D. tertiolecta
showed a positive
response on cell growth in terms of cell density to
increase of temperature. Figure 1(a) showed that there
was a significant decrease in cell density of
D.
tertiolecta
to increase temperature. This result was
supported by Garcia et al (2007) who found that
growth of
D. salina
and
D. viridis
decreased significantly
with increasing temperature.