1231
A publication of

CHEMICAL ENGINEERING TRANSACTIONS
VOL. 106, 2023

The Italian Associationof Chemical Engineering

Online at www.cetjournal.it

Guest Editors: Jeng Shiun Lim, Nor Alafiza Yunus, Peck Loo Kiew, Hon Huin Chin
Copyright © 2023, AIDIC Servizi S.r.l.
ISBN 979-12-81206-05-2; ISSN 2283-9216

DOI: 10.3303/CET23106206

Effect of Agitation on Growth and Fatty Acid Composition ofGreen Microalgae Acutodesmus obliquus Q2-12E
Nur Faiqah Amzaha,b, Shaza Eva Binti Mohamada,*, Fazrena Nadia Md Akhira, Fatin
Syahirah Othmana, Iwane Suzukib, Yoshiaki Maedab
Department of Chemical and Environmental Engineering, Malaysia-Japan International Institute of Technology, Universiti
Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100 Kuala Lumpur, MalaysiabFaculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8527, Japanshaza@utm.myaDifferent environmental factors including agitation can affect the growth of microalgae and fatty acidcomposition. It is necessary to agitate the culture to ensure that each cell received enough amount of nutrition,
air, and light exposure. This study aimed to analyze the growth and fatty acid composition of Acutodesmus
obliquus Q2-12E cultivated with molecular diffusion (no agitation) and shaking and compare those with aprevious study employing the aeration method. The results showed that in comparison to molecular diffusion,agitation by shaking and aeration on the culture significantly increased the specific growth rate. Culture underaeration was revealed to exhibit the fastest growth rate (0.1444 day-1) compared to shaking (0.0951 day-1) andmolecular diffusion (0.0614 day-1). The lipid obtained using modified Folch’s method was esterified and analyzed
using gas chromatography with a flame-ionization detector (GC-FID) with heptadecanoic acid (C17:0) as theinternal standard. The analyzed result shows that the major fatty acids produced both by molecular diffusionand shaking method were monounsaturated fatty acids which were 39.5% and 46%, respectively. While themajor fatty acid produced by the aeration method was polyunsaturated fatty acid (38.6%). Molecular diffusionand shaking could not produce eicosatetraenoic acid (C20:4) which was detected with the aeration method.
This study demonstrated that the same microalga strain exhibited a different growth and fatty acid composition
depending on the agitation methods.

1. Introduction
Microalgae have received a lot of interest recently because of their potential advantages as a bioresource thatmight be utilized for a variety of goods, including fish feed, human food, pharmaceuticals, and different sourcesof feedstock for the upcoming generation of biofuels (Kamyab et al., 2019). It has been considered an essential
resource to fulfill the global demand for sustainable energy and green technologies. Acutodesmus obliquus (alsoknown as Scenesdesmus obliquus or Tetradesmus obliquus) (Oliveira et al., 2021) is one of the most prevalent
species of green microalgae in freshwater ecosystems. It is one of the oleaginous microalgae which has been
identified as an excellent freshwater oleaginous microalgal species for research because of its rapid growth inlarge quantities, ease of cultivation, strong resistance to bacterial infections, and its ability to adapt to a varietyof environmental conditions (Othman et al., 2019). Many environmental factors, including temperature, pH,
oxygen content, salinity, and light intensity, can have an impact on the growth of microalgae and the biochemicalor physiological characteristics of microalgal cells (Ajala & Alexander, 2022). Because culture conditions differ
from species to species, all of these growth variables must be specified for successful microalgae cultivation for
a particular application (Chowdury et al., 2020). Another factor that may affect some microalgae's growth rates
and fatty acid profile is agitation. The frequency with which cells are exposed to light and dark volumes as wellas the amount of mass transferred between the cell and the nutrients will all be improved by effective mixing.
The culture grown in flasks can be mixed in a variety of ways. For instance, it can be manually mixed byhandshaking and bubbling utilizing aeration. Another type of mixing is orbital shaking which is caused by theorbital displacement of a vessel and provides a rotating path with a set angular velocity as well as a permanent
Paper Received: 05 June 2023; Revised: 15 July 2023; Accepted: 22 September 2023
Please cite this article as: Amzah N.F., Mohamad S.E.B., Akhir F.N.M., Othman F.S., Suzuki I., Maeda Y., 2023, Effect of Agitation on Growthand Fatty Acid Composition of Green Microalgae Acutodesmus obliquus Q2-12E, Chemical Engineering Transactions, 106, 1231-1236
DOI:10.3303/CET23106206

1232

direction for an inertial force. It is advised to use orbital shaking to maintain a low-shear stress environment andavoid cellular damage in active fluid suspension (Samadi et al., 2022). The high rates of aeration flows cancause hydrodynamic stress that impacts growth, cell damage, and finally cell death while maintaining a highlevel of mixing and incurring higher energy and carbon dioxide costs. Agitation's impact on microalgal growthand biochemical parameters is influenced by both the algal strain and the culture conditions such as themedium's ionic strength, salinity, temperature, and nutrition. Besides, microalgae growth and biofiltration are notdependent on aeration per se in cases where nutrient concentrations are high enough (Mansouri et al., 2022).
In this present study, the effect of agitation on microalgae growth and fatty acid composition in A. obliquus Q212E was analyzed and compared with a previous study employing aeration with carbon dioxide supplied.

2. Materials and methods
2.1 Microalgae strain and culture conditions
A.obliquus (Q2-12E, MJIIT-UTM, Malaysia), a green microalga was used in this study. It was initially studied byresearchers from Algal Biomass Research Lab, Malaysian-Japan International Institute Technology (MJIIT),
Universiti Teknologi Malaysia (UTM) for the production of omega-3 fatty acids. Before conducting theexperiment, stock culture was prepared with inoculum size in the culture is 10% (v/v) and the cultivation for themain experiment was performed using the batch of culture in the exponential growth phase. Cells were cultivatedin no agitation and agitation methods which were molecular molecular diffusion and shaking. 10 % (v/v) of stock
culture was cultivated in Erlenmeyer flasks with 100 mL of the modified AF6 medium under sterile conditions.
The pH of the medium was adjusted to pH 6.6. Microalgal cells were grown for a 12h photoperiod at 25°C undera light intensity of approximately 80 µmol photons/m2/s. For molecular diffusion mode, the Erlenmeyer flasks
were placed standstill without agitation. In the shaking mode, cells were agitated at 140 rpm using TAITEC
double shaker NR-30. The microalgae were cultivated for 14 days in the growth chamber until early stationary
growth phase. Meanwhile, from previous study of aeration mode, 2% of CO2 was supplied.
2.2 Growth of microalgae
The growth of the microalgae was studied by plotting the growth curve and monitored until the death phase.
Microalga culture was incubated at an optimum temperature under light sources for 14 days. Microalgae growth
was monitored at 750 nm of optical density (OD750nm) with an ultraviolet-visible (UV-Vis) spectrophotometer(UV-1900, Shimadzu) on an alternate day. The specific growth rate (µ, day-1) was to evaluate the rate ofincreasing cell number per time during the exponential growth phase by using Eq (1).
𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔ℎ 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 (µ, 𝑑𝑑𝑑𝑑𝑑𝑑 −1 ) =𝑙𝑙𝑙𝑙𝑋𝑋2 − 𝑙𝑙𝑙𝑙𝑋𝑋1
𝑡𝑡2 − 𝑡𝑡1

(1)

Where 𝑋𝑋1 , 𝑋𝑋2 , 𝑡𝑡1 , and 𝑡𝑡2 are OD750nm value at the early exponential growth phase, OD750nm value at the late
exponential growth phase, and day corresponding to 𝑋𝑋1 , and the day corresponding to 𝑋𝑋2.
The doubling time of the culture is calculated to determine the number of cell divisions per day based on thevalue of the specific growth rate using Eq (2).
𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡, 𝑡𝑡𝑑𝑑 (𝑑𝑑𝑑𝑑𝑑𝑑) =

𝑙𝑙𝑙𝑙2
µ

(2)

2.3 Fatty acid methyl esters (FAME) extraction
Following the extraction of the lipid content, the lipid undergoes a transesterification process to produce the
FAMEs of this three-agitation method. Centrifugation was used to harvest 100 mL of the microalgae culture for10 min at 25°C at 12, 000 x g. using High Speed Refrigerated Centrifuge (SRX 201, TOMY). Prior freeze-drying
for 3 days using a freeze dryer (FDU-1100, EYELA), the microalgae pellet was frozen at -80°C for one day. The
total lipid was extracted using chloroform-methanol solvent-based Folch’s method (Folch et al., 1957) with somemodifications. The dry lipid obtained were weighed using a weighing scale and expressed in percentage of dry
cell weight. Dry lipid samples were treated with 4 mL of 0.1N methanolic: hydrochloric acid (MeOH: HCl) andleft in a water bath at 100°C for 1 h. 4 mL of hexane was added to the cooled mixture. Then, the mixture wasshaken vigorously to form a double layer. The upper layer of the mixture was transferred to a new extraction
tube. 2 mL of distilled water and 2 mL of hexane are added to the remaining layer. Again, the upper layer wastransferred to the first upper layer. The mixture was dried using a rotary evaporator for 30 min using a centrifugal
concentrator (CC-105, TOMY). The dry lipid samples were dissolved in hexane and transferred to a glass vial
for gas chromatography analysis.

1233

2.4 Gas chromatography analysis
After reaching the stationary phase, FAs were collected and analyzed by Gas chromatography with a flameionization detector (GC-FID) to determine the FA composition. GC-FID: GC-2014, Shimadzu, Japan and Agilent
CP-Sil 5 CB column (50 m x 0.32 mm x 0.12 µm) was used to measure and classify the FAMEs profile. Helium
was used as carrier gas with a constant flow rate in splitless mode. The initial temperature was set to 60 °C for
1.5 min then increased to 130 °C at 20 °C/min. Then, the temperature increased to 250 °C at 4 °C/min. 1 µL of
the sample was injected into GC. By comparing the retention times of the peaks of known FAMEs in the standard
solution (FIM-FAME-7 Mixture, C4:0-C22:6, Matreya) to the retention times of the FAMEs in the microalgal lipid
sample, the FAMEs in the sample were identified. By comparing the total FAME chromatogram's peak area tothe peak area of the internal standard, the amount of FAMEs was determined (C17:0) using Eq (3):
𝐴𝐴𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹
𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐, % = �� 𝑥𝑥 100%∑ 𝐴𝐴 − 𝐴𝐴𝐶𝐶17(3)

Where 𝐴𝐴𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 , ∑ 𝐴𝐴, 𝐴𝐴𝐶𝐶17 are the peak area of FAME, the total peak area of the FAMES chromatogram, and theinternal standard peak area (C17:0).

3. Result and discussion
3.1 Effect of agitation on cell growth

OD 750 nm

The growth curve of A. obliquus Q2-12E with different agitation modes was constructed by monitoring the
OD750nm every alternate day within 14 days period. In the microalgal growth curve, there are four stages. The
first stage is the lag phase, where the microalgae undergo physical adjustments to adapt to the nutrition in the
new medium. The second stage will be the exponential phase, in which microalgae rapidly consume thenutrients in the culture medium and the amount of biomass is expected will increase significantly. The next stage
is the stationary phase, in which there will be a nutrient limitation and the growth and death of microalgae at thesame rate. Finally, the death phase will be the last stage of the growth curve where the microalgae are unable
to grow due to nutrient depletion and pH disturbance (Thong et al., 2022). Based on Figure 1, the growth profile
of A. obliquus Q2-12E cultivated under aeration + CO2 was found to be higher compared to molecular diffusionand shaking mode. Algal growth was limited by CO2 lower than 5% because the cell was unable to access the
carbon requirement needed for full cell growth. Besides, cells that were cultivated under the shaking andaeration method entered the exponential phase on day 2 of cultivation, while the molecular diffusion method
entered the exponential phase on day 4. The quantity of nutrients and light made available to cells by their
spatial displacement and homogeneity is the reason why the exponential phase of agitation cultures begins
more quickly compared to molecular diffusion (Samadi et al., 2022). At the stationary phase, molecular diffusion
entered the phase later than the culture under shaking and aeration which were on days 12, 8, and 6. Comparedto the stationary cultures, efficient mixing within the batches permitted the cell to effectively absorb nutrientsfrom uncharted areas and be exposed to light.
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

Molecular Diffusion
Shaking
Aeration + CO2

0

2

4

6

8

10

12

14

Time of cultivation (day)Figure 1: Growth curve of A. obliquus Q2-12E for molecular diffusion (blue circles), shaking (red circles),andaeration+CO2 (green circles). Data for aeration+CO2 was obtained in the previous study
Figures 2 show the specific growth rate of A. obliquus Q2-12E under three different agitation modes during the
exponential phase. The specific growth rate assessed an microorganism's ability to grow by counting how many

1234

times it could reproduce over time. The result demonstrates that a specific growth rate of A. obliquus Q2-12E
differed significantly under all agitation modes. There are various ways that agitation promotes the growth of
microalgae. It prevents the cell from self-shading, breaches the boundary layer in the culture system, and makes
it easier for cells to absorb CO2, nutrients, and light (Samadi et al., 2022). Figure 2 shows that the specificgrowth rate of cultures cultivated by shaking and aeration modes was higher, which were 0.1444 day-1 and
0.0951 day-1 compared to cultures grown in molecular diffusion (0.0614 day-1) method. Nevertheless, thespecific growth rate for aeration was slightly higher than shaking which indicates that the aeration+CO2 wasmuch more effective to agitate the culture. CO2 concentration plays various important roles for microalgae, oneof which is photosynthesis. On supplementation of CO2, it facilitates the specific growth rate of cells in aeration
mode by improving the photosynthetic activity and different photosynthetic pigment patterns. Besides, the resultshows that due to nitrogen and phosphorus limitation and no supplementation of CO2 give an extreme impact
on algal growth for both modes, with no agitation and agitation (Kumari et al., 2021). In addition, according tothese findings, agitation has an important impact on the interaction with other microalga growth conditions.

Specific Growth rate(day-1)

0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0

Molecular
diffusion

Shaking

Aeration

Agitation method

Figure 2: Specific growth rate of A. obliquus Q2-12E for three different agitations. Data for aeration+CO2 was
obtained in the previous study
3.2 Effect of agitation on fatty acid composition
Microalgal cultivation has a noticeable effect on fatty acids (FAs) composition, which is also useful as abiochemical marker of cultivation differences. In this study, the FAMEs profile of the culture under three agitation
modes was examined and compared to determine the type of FAs produced by A. obliquus Q2-12E with adifferent agitation mode. Table 1 depicts the major FA composition detected in different agitation modes. Thesefindings revealed that the molecular diffusion mode has a higher content of saturated fatty acids (SFA) comparedto the other two agitation modes. Meanwhile, monounsaturated fatty acid (MUFA) was the highest under shaking
mode which was at 46 %. It can be deduced that under aeration+CO2 have the highest concentrations ofpolyunsaturated fatty acid (PUFA), which was 38.6 %. Meanwhile, the PUFA percentage under molecular
diffusion and shaking has not much different which were 24.1 % and 20.5 %. According to the result, theproduction of PUFA by A. obliquus Q2-12E was more favorable under aeration with supplementation of CO2.
CO2 concentration facilitates the accumulation of total lipids and PUFAs in microalgae. The greater PUFA
content in cells with CO2 supply may have resulted from the inhibition of FA synthesis, which encourages thedesaturation of existing FAs (Liang et al., 2020).
Table 1: The major fatty acid composition (%) of A. obliquus Q2-12E under three different agitation modes
Types of fatty acids

Percentage to total fatty acids (%)
Molecular diffusion
Shaking
SFA
36.5
33.5
MUFA
39.5
46.0
PUFA
24.1
20.5
Note: Data of aeration+CO2 was obtained in the previous study

Aeration + CO2
26.0
35.4
38.6

1235

Table 2 shows the fatty acid profile of A. obliquus Q2-12E varied under different agitation modes. In this study,
a total of 9 and 11 FAs were detected by A. obliquus Q2-12E under molecular diffusion and shaking,respectively. While, under aeration+CO2, five FAs were found. The shares of FA C16:1 and C20:3 in total fatty
acid (%) were found low (<1 %) under molecular diffusion mode. While, under shaking mode, the shares of FA
C14:0, C14:1, and C20:3 was relatively low (<1 %). Stearic acid (C18:0), which occurs most frequently in living
things, was the predominant SFA in all agitation modes. Oleic acid (C18:1 cis-9) was the most MUFA detectedin this study for molecular diffusion and shaking method which was 36.20 % and 41.28 %. However, no C18:1
cis-9 was detected for aeration+CO2. The untreated microalgae with CO2 profile are excellent for biodiesel
synthesis since the fatty acid profiles high in SFAs and MUFAs are the most suitable for those applications. A.
obliquus Q2-12E under those cultivation conditions has the potential to be one of the microalgae that areimportant for biofuel feedstock for the future. The primary components of the oil produced by microalgae are
typically unsaturated fatty acids, mainly palmitoleic (16:1), oleic (18:1), linoleic (18:2), and linolenic acid (18:3),
with only a minor amount of palmitic (16:0), SFAs percentage derived from saturated stearic acids (C18:0)(Rahman et al., 2019). Acutodesmus sp. was known as α-Linolenic acid (C18:3) producing microalgae (Othman
et al., 2019), however, surprisingly under aeration+CO2 there was no C18:3 detected. Under molecular diffusionand shaking mode, the amount of C18:3 detected was relatively similar which was 8.98 % and 8.10 %,respectively. Interestingly, only aeration+CO2 can generate eicosatetraenoic acid (C20:4). The addition of CO2
into the culture has proven to elevate the omega-3 FAs content or synthesis in the microalgae (Barati et al.,2022). In addition, the approach of investigating novel microalgal strains as a potential source of ETA has
generated scientific interest. This is due to persistent concerns about the increased demand for high-purity
omega-3 fatty acids, specifically ETA, from the growing global population (Mohamad et al., 2021). An increasein ETA consumption can lead to enhanced overall health and well-being, hence effective strategy to boost theamount of ETA in this strain should be conducted.
Table 2: The fatty acid (FA) profile of A. obliquus Q2-12E with different agitation modes
Type

Saturated

Fatty acids

Common name

C14:0
Myristic acid
C16:0
Palmitic acid
C18:0
Stearic acid
Monounsaturated
C14:1
Myristoleic acid
C16:1
Palmitoleic acid
C18:1 trans-9
Elaidic acid
C18:1 cis-9
Oleic acid
C20:1
Eicosenoic acid
Polyunsaturated
C16:4
Hexadecatetraenoic acidC18:2 cis-9
Linoleic acidC18:3
α-Linolenic acidC18:2 trans-9
Linoelaidic acid
C20:3
Eicosatrienoic acid
C20:4
Eicosatetraenoic acid
Note: Data of aeration+CO2 was obtained in the previous study

Percentage to total acids (%)
Molecular
Shaking
Aeration+CO2
diffusion
ND
0.05
22.11
35.07
32.14
ND
1.57
1.49
26.04
ND
0.18
ND
0.63
2.83
ND
2.82
1.95
ND
36.20
41.28
ND
ND
ND
13.39
ND
ND
22.09
11.58
9.93
ND
8.98
8.10
ND
3.22
2.26
ND
0.42
0.29
ND
ND
ND
16.65

4. Conclusions
The effect of the agitation method of A. obliquus Q2-12E on growth and fatty acid composition was determined
and compared with the previous study by aeration with 2% CO2. The result shows that agitation of culture canenhance the specific growth rate of the microalga compared to the motionless method. Agitation by aeration
with supplementation of CO2 depicts to be more efficient on the cell growth followed by shaking mode andmolecular diffusion. This difference can be attributable to the two mixing types' different distributions of wall
shear stress and fluid motion. The culture's nutritional uptake, as well as the uniformity of the light andtemperature distribution, are all improved by agitation. Besides, supplementation of CO2 had a notable impact
on the FAs profiles which, the PUFA content was significantly increased and rare ETA was able to be produced.
Nevertheless, without supplementation of CO2, the cells are able to produce an interesting amount of MUFAs
and SFAs for biofuel production. These findings demonstrated that agitation interacts with other culture medium
variables in significant ways, and that optimization is necessary for microalgal cultivation systems to scale up
effectively.

1236

Acknowledgments
The authors are grateful for funding from Majlis Amanah Rakyat (MARA) and special thanks to the MalaysianJapan International Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur, and the University ofTsukuba, Japan for the equipment and facilities provided.
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