For
microalgae, it is essential to choose the right species in order to be useful
aquaculture species. Chaetoceros sp., Pavlova lutheri, Isochrysis sp. (T.ISO),
Tetraselmis suecica, Skeletonema costatom and Thalassiosira pseudonana have
been found to be as the microalgae that contain good nutritional value either
as monospecies or in a mixed diet (Enright et al., 1986b; Thompson et al.,
1993; Brown et al., 1997).
Generally,
the growth of microalgae is characterized by five stages. Monitoring the growth
is very essential for maintaining healthy culture and avoiding organic sedimentation.
Algae would grow optimally when the nutrients and light source are sufficient.
It is an average of 0.07 densities increasing day by day and would reach 0.8 OD
on stationary phase. The algae will dead after the stationary phase and the debris
will accumulate in ponds. This will make an issue of low productivity, low
quality and bad odor during the process. We have to filter the algal debris
when it is reaching 3rd phase or 0.8 OD.
There
is numerous direct and indirect methods used to determine progressive growth in
microalgae cultivation. Direct methods are algal biomass, packed cell volume,
cell counts and detecting pigment contents. Indirect methods are primary
productivity and changes in chemistry of the aqueous environment used to
express algal growth quantitatively.
OBJECTIVES
This experiment is done mainly to determine the microalgal growth through different ways of measurements which are estimation of individual dry weight, optical density reading and chlorophyll analysis.
METHODOLOGY
A) Estimation
of individual dry weight
Dry weight of algal cells can be determined by
filtering and drying algae form aliquots of culture of known concentration.
1. The
concentration of the algal culture [N] to be sampled for dry weight analysis is
determined accuratedly (3 duplicate counts).
2. An
exact volume [V] on pretared glass microfiber filters [W] is filtered using a
Buncher setup connected to a vacuum pump (in triplicate).
3. The
filter is washed with a solution of ammonium formate (0,5M) to remove salts.
4. The
same procedure is followed with control filters on wich equal volume of 0.22 µm
filtered seawater is filtered (in triplicate) [B].
5. The
filter is dried at 100oC for 4 hour to volatilize the ammonium
formate.
6. Weight
is taken on an analytical balanced [W*-B*]
7. The
dry weight per algal cell is calculated according to the formula :
DW (g/cell) = DWWC
– DWBC
N
X V
8. For
algal species dry weight per cell is expressed in pg (pictogram) per cell.
A) Optical
density reading
1. Microalgal
culture solution was diluted with seawater at 100%, 80%, 60%, 40%, 20% and 0 in
the test tubes.
2. The
absorption of this series of dilution was measured with the spectrophotometer
and the values were calculated.
A) Chlorophyll
analysis
1. 50ml
of sample was filtered through GF/F filtered.
2. 3
to 5 drops of MgCO3 was added into the sample as it was being
filtered.
3. The
edges of the filter which are not coated with residue was trimmed away.
4. The
filter was homogenized with 5ml of acetone for 1 minute. Then 5ml more of acetone
was added and being grinded for 30 seconds.
5. The
sample extract was put in refrigerator in the dark for30 minutes.
6. Then,
the samples were centrifudged at 3000rpm for 10 minutes.
7. The
absorbance of the sample extracts was measured at 750nm, 664nm, 647nm and
630nm.
8. The
chlorophyll amount was calculated by using the equation as below:
Ca
= 11.8(664nm – 750nm) – 1.54(647nm – 750nm) – 0.08(630nm – 750nm)
Where;
A
= Acetone extract in ml
S
= Volume sample filtered in ml
Figure 1: Acetone extraction
Figure 2: Extraction in 1ml tube
Figure 3: Extraction in plate
A) Estimation
of individual dry weight
|
Volume
[V]
|
20
ml
|
|
Conc.
[N]
|
5.17
x 107
|
|
Blank
|
B
|
B*
|
BC = B* - B
|
|
|
1
|
0.0950
|
0.0982
|
0.0032
|
|
|
2
|
0.0943
|
0.0976
|
0.0033
|
|
|
3
|
0.0952
|
0.0987
|
0.0035
|
|
|
Average
|
|
|
0.0033
|
|
|
|
|
|
|
|
|
Sample
|
W
|
W*
|
WC = (W*-W)-Bc
|
Pg/cell
|
|
1
|
0.0950
|
0.1585
|
0.0602
|
5.50
x 10-11
|
|
2
|
0.0943
|
0.1989
|
0.1013
|
9.48
x 10-11
|
|
3
|
0.0952
|
0.1943
|
0.0958
|
8.95
x 10-11
|
|
|
|
|
|
|
|
|
|
|
|
|
A) Optical
density reading
|
Dilution
(%)
|
Haemocytometer
count (cell)
|
|
0
|
0
|
|
20
|
220,000
|
|
40
|
386,500
|
|
60
|
1,875,000
|
|
80
|
1,928,333
|
|
100
|
2,761,500
|
A) Chlorophyll
analysis
|
BlankSubtraction1
|
||||||
|
Parameters
|
||||||
|
Blank
type
|
plate
blank
|
|||||
|
Plate
1: Plate 1 - Wavelength: 630 nm
|
||||||
|
Abs
|
1
|
2
|
3
|
4
|
5
|
6
|
|
A
|
0.0399
|
0.0308
|
0.0599
|
0.1075
|
0.1544
|
0.2095
|
|
B
|
0.0363
|
0.0612
|
0.0657
|
0.1648
|
0.1668
|
0.2154
|
|
C
|
0.0749
|
0.0979
|
0.1070
|
0.1560
|
0.2052
|
0.2521
|
|
D
|
||||||
|
E
|
0.1258
|
0.1292
|
-0.0008
|
0.0032
|
-0.0004
|
0
|
|
F
|
0.0834
|
0.0866
|
0.0815
|
0.0888
|
0.0947
|
|
|
G
|
0.0932
|
0.1005
|
0.1190
|
0.1133
|
0.0961
|
|
|
H
|
||||||
|
Plate
1: Plate 1 - Wavelength: 647 nm
|
||||||
|
Abs
|
1
|
2
|
3
|
4
|
5
|
6
|
|
A
|
0.0365
|
0.0334
|
0.0671
|
0.1200
|
0.1724
|
0.2327
|
|
B
|
0.0343
|
0.0647
|
0.0735
|
0.1768
|
0.1882
|
0.2400
|
|
C
|
0.0747
|
0.1003
|
0.1144
|
0.1700
|
0.2244
|
0.2768
|
|
D
|
||||||
|
E
|
0.2459
|
0.2510
|
-0.0009
|
0.0029
|
-0.0008
|
0
|
|
F
|
0.1562
|
0.1655
|
0.1505
|
0.1693
|
0.1807
|
|
|
G
|
0.1267
|
0.1372
|
0.1551
|
0.1518
|
0.1320
|
|
|
H
|
||||||
|
Plate
1: Plate 1 - Wavelength: 664 nm
|
||||||
|
Abs
|
1
|
2
|
3
|
4
|
5
|
6
|
|
A
|
0.0359
|
0.0384
|
0.0776
|
0.1399
|
0.2022
|
0.2721
|
|
B
|
0.0322
|
0.0720
|
0.0827
|
0.1962
|
0.2164
|
0.2771
|
|
C
|
0.0732
|
0.1063
|
0.1259
|
0.1932
|
0.2549
|
0.3155
|
|
D
|
||||||
|
E
|
0.5609
|
0.5747
|
-0.0023
|
0.0023
|
-0.0009
|
0
|
|
F
|
0.3392
|
0.3668
|
0.3248
|
0.3754
|
0.3978
|
|
|
G
|
0.2192
|
0.2360
|
0.2571
|
0.2549
|
0.2280
|
|
|
H
|
||||||
|
Plate
1: Plate 1 - Wavelength: 670 nm
|
||||||
|
Abs
|
1
|
2
|
3
|
4
|
5
|
6
|
|
A
|
0.0340
|
0.0424
|
0.0866
|
0.1571
|
0.2283
|
0.3031
|
|
B
|
0.0319
|
0.0766
|
0.0927
|
0.2132
|
0.2432
|
0.3095
|
|
C
|
0.0730
|
0.1111
|
0.1360
|
0.2094
|
0.2809
|
0.3466
|
|
D
|
||||||
|
E
|
0.4213
|
0.4327
|
-0.0029
|
0.0010
|
-0.0021
|
0
|
|
F
|
0.2527
|
0.2740
|
0.2417
|
0.2769
|
0.2945
|
|
|
G
|
0.1796
|
0.1900
|
0.2104
|
0.2084
|
0.1841
|
|
|
H
|
||||||
|
Plate
1: Plate 1 - Wavelength: 750 nm
|
||||||
|
Abs
|
1
|
2
|
3
|
4
|
5
|
6
|
|
A
|
0.0289
|
0.0198
|
0.0409
|
0.0793
|
0.1123
|
0.1549
|
|
B
|
0.0322
|
0.0507
|
0.0536
|
0.1306
|
0.1292
|
0.1679
|
|
C
|
0.0713
|
0.0875
|
0.0951
|
0.1319
|
0.1705
|
0.2057
|
|
D
|
||||||
|
E
|
-0.0079
|
-0.0062
|
-0.0072
|
-0.0045
|
-0.0087
|
0
|
|
F
|
0.0094
|
0.0048
|
0.0112
|
0.0059
|
0.0098
|
|
|
G
|
0.0526
|
0.0569
|
0.0713
|
0.0677
|
0.0553
|
|
Table 3: Absorbance of sample reading (row F)
|
Sample
|
1
|
2
|
3
|
4
|
5
|
|
Ca
|
3.6596
|
3.9706
|
3.4987
|
4.0662
|
4.3125
|
|
|
|
|
|
Average
|
3.9015
|
|
|
|
|
|
Chlorophyll
a (mg/L)
|
7.803-5
|
DISCUSSION
Based
on Table 1, it shows that the dry weight per cell of the microalgae is too
little which are 5.50 x 10-11, 9.48 x 10-11 and 8.95 x 10-11.
This can be said that the culture solution is not bloomed yet which mean there
is not too much cell in the solution. This situation is maybe caused by whether
the stock solution is still new and its phase do not reach exponential stage. Besides
that, it can also be said that the microalgae already in death phase that is
why there is low amount in cell count.
Meanwhile
based on Table 4, the sample was made into 5 replicates. As an average, the Ca
value for the sample is 3.9015. After being calculated by using the given
formula, the chlorophyll value for the sample is 7.803-5. Measuring
the concentration of chlorophyll a is much easier than counting algal cells and
provides a reasonable estimate of how much algae is in the water. Chlorophyll a
is measured because it is the most common type of chlorophyll—the green pigment
that is responsible for a plant's ability to convert sunlight into usable
energy. It is usually the parameter used as the trophic indicator, mainly
because the relationship between the content of this pigment and the amount of
algal biomass is quite direct. Louda and Monghkonsri [7] compared
spectrophotometric estimates of chlorophyll contents with those obtained by
high performance liquid chromatography (HPLC). They concluded that spectrophotometric
evaluation of chlorophyll, using UNESCO [8] and Jeffrey and Humphrey [9] equations,
gave excellent results. Those authors support that in the absence of
significant differences between the two referred methods, the
spectrophotometric analyses are much less expensive and much faster than HPLC
analyses, making them a good tool for routine chlorophyll evaluation. Methanol
and ethanol compare well as extraction solvents used in chlorophyll evaluation
from microalga biomass. Both of them showed to be better than acetone, which is
not very efficient in the extraction and quantification of pigments from
autotrophic cell cultures.
CONCLUSION
There
is numerous direct and indirect methods used to define progressive growth in
microalgae cultivation. Direct methods are dry weight, packed cell volume, cell
counts and detecting pigment contents. Indirect methods are primary productivity
and changes in chemistry of the aqueous environment used to express algal
growth quantitatively.
REFERENCES
Brown, M. R. (2002). Nutritional value and use of microalgae in aquaculture. Avances en Nutrición Acuícola VI. Memorias del VI Simposium Internacional de Nutrición Acuícola, 3, 281-292.
Mirón, A. S., Garcıa, M. C. C., Camacho, F. G., Grima, E. M., & Chisti, Y. (2002). Growth and biochemical characterization of microalgal biomass produced in bubble column and airlift photobioreactors: studies in fed-batch culture. Enzyme and Microbial Technology, 31(7), 1015-1023.
Ra, S., & Rajendranb, S. Growth measurement technique of microalgae.
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