Basic HTML Version
International Journal of Marine Science 2013, Vol.3, No.17, 135-144
http://ijms.sophiapublisher.com
137
0.5 g NaNO
3
, 0.25 g CaCl
2
.2H
2
O, 0.2 g tris-base,
0.165 g NaHCO
3
, 1.0 mL of 3% solution KH
2
PO
4
and
6.0 mL of trace elemental solution. The trace elemental
solution (per liter) includes 0.02 g CuSO
4
.5H
2
O, 0.0125
g NaMO
4
.2H
2
O, 9.0 g Fe citrate, 9.0 g Citric acid,
0.046 g ZnSO
4
.7H
2
O, 0.289 g MnCl
2
.4H
2
O, 0.0081 g
CoCl
2
.6H
2
O, 0.1001 g FeSO
4
.7H
2
O, 2.0027 g EDTA,
2.2877 g H
3
BO
3
, 0.010 g vitamin B
12
, 0.005 g Biotin
and 0.02 g Thiamine HCl
. Scenedesmus
sp. is
freshwater microalgal was cultured in MLA medium,
consisting of 2 L pasteurized distillated water which
has the following composition (per liter): 49.4 g
MgSO
4
.7H
2
O, 85 g NaNO
3
, 6.96 g K
2
HPO
4,
2.47 g
H
3
BO
3
, 0.00129 g H
2
SeO
3
, 16.9 g NaHCO
3
, 29.4 g
CaCl
2
.2H
2
O, 10 mL micronutrients. The micronutrient
solutions (per liter) includes 4.36 g Na
2
EDTA, 1.58 g
FeCl
3
.6H
2
O, 0.6 g NaHCO
3
, 0.36 g MnCl
2
.4H
2
O, 1.0
g CuSO
4
.5H
2
O, 2.2 g ZnSO
4
.7H
2
O, 1.0 g CoCl
2
.6H
2
O,
0.6 g Na
2
MoO
4
.2H
2
O, 0.010 g Biotin, 0.010 vitamin
B
12
and 0.010 g Thiamine HCl. For the treatments of
N and P depletion, the culture media were not added
of N and P elements, such as NaNO
3
and K
2
HPO
4
.
2.2 Culture system
Growth experiments were done at different temperatures
and nutrient conditions in 500 mL-Erlenmeyer flasks.
The medium and flasks were sterilized in an
autoclave for 20 mins at 121
℃
in order to prevent
any contamination during the early stages of growth.
The cell cultured was kept at incubator room at 18
℃
and 25
℃
equipped with artificial lightening. Each
autotrophic batch cultivation was carried out in
triplicate for 9 and 10 days at a continuous photon
flux density of 180 µmol m
-2
s
-1
, which was measured
by a light intensity meter (LICOR Model LI-1400
data logger) for 24 hours.
Temperature and nutrient condition in the medium
were selected as a treatments (independent variables).
Two different temperatures condition were selected,
such as 18
℃
and 25
℃
, while three different nutrient
conditions were control (deplete nutrient medium),
nitrogen and phosphate depletion medium.
2.3 Microalgal cell counting and dry weight
A direct microscopic cell count was performed daily
with Brightline Hemocytometer (Neubauer, Weber
England) and a Olympus CHS model microscope
(Olymphus Optical Co. Ltd, Japan).
Algae were cultivated in flasks for biomass determination.
10 mL samples were filtered on to pre-dried and
weighed GF/C fibre filters every second day of culture.
Filters were oven dried overnight at 60
℃
, and
reweighed using an analytical scale.
2.4 Measurement of growth rate
Specific growth rate (µ d
-1
) was calculated as follows:
µ=In(Wt/Wo)/
△
t, where Wt and Wo were the final and
initial biomass concentration, respectively.
△
t was the
cultivation time in day (Ono and Cuello, 2007).
2.5 Measurement of chlorophyll
a
The measurement of chl
a
was taken every second day
of cell cultivation. Sample (10 mL) of the culture was
filtered using a 25 mm GF/C filter. Filtered cell was
placed into 10 ml centrifuge tube and 9 mL of 100%
cold acetone was added. The tubes were wrapped in
foil and placed at a fridge overnight for extracting
chlorophyll. The next day, the samples were sonicated
with probe sonicator (Bronson Sonifier 450, Unisonic,
Australia) for 3 min and adding 1 mL dH
2
0. Samples
were centrifuged (Heraeus Multifuge 3SR, Thermo
Scientific, Australia) at 3000 rpm for 10 min.
Chlorophyll a concentration (µg/mL) was determined
using Spectrophotometer (Cary 50 Bio UV-Visible
Spectrophotometer) with the wavelength of 630, 647,
and 750. The relationship of the Chl
a
amount in
supernatant with those wavelength was correlated
according to Jeffrey and Humphrey (1975): Chlorophyll
a=11.85E
664
–1.54E
647
–0.08E
630
.
2.6 Lipid analysis
2.6.1 Nile Red Fluorescence assay
A fluorescence spectrometric method was applied for
fast determination of lipid content using a Hitachi
F-2000 Fluorometer. Measurements were recorded
daily. In the method, the microalgal cells were stained
with Nile Red (Sigma, St.Louis, MO, USA) followed
the protocol reported by Elsey et al (2007). In brief, 3
mL of culture was pipetted into a glass cuvette. A
fluorescence measurement as a standard culture was
noted. Excitation wavelength was set at 486 nm and
the emission wavelength was 570 nm. Fluorescence
intensity was recorded over a period of 30 seconds
and the result of fluorescence intensity reading vs time
was displayed as a graph using a Toshiba Satellite
4080XCDT laptop computer with a software program
of Logger Pro. The average of fluorescence intensity