Jurnal Airaha, Vol.10, No.

02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638

The Growth of Chlorella sp. With Varying Nutrient Concentration

Intanurfemi B. Hismayasari*, Ernawati, Agung Setia Abadi,

Asthervina Widyastami Puspitasari
Politeknik Kelautan dan Perikanan Sorong
*Corespondensi : ib.hismayasari@gmail.com

Received : November 2021 Accepted : December 2021

ABSTRACT
The aim of this study was to determine the effect of differences addition of culture medium
concentrations on the growth of microalgae type Chlorella sp. Samples were obtained from
the Center for Brackish Water Cultivation Fisheries (BBPBAP), Jepara. This research was
conducted at the Laboratory of Nutrition and Natural Feed Sorong Marine and Fisheries
Polytechnic with an experimental method using four treatments with addition of walne
medium, there were A (adding only at the beginning of cultivation); treatment B (adding
every day); C (adding every two days), and treatment; D (adding every three days). The cell
growth performance was significantly found in treatment C with the addition of nutrients
every two days with a peak cell density of Chlorella sp was 2,628,450 cells / ml. There is a
need for mass culture assay to determine the level of effectiveness and efficiency.
Keywords: Chlorella sp.; growth performance; natural food

INTRODUCTION conditions for microalgal cultures are strain

Chlorella sp. is a green alga used as
natural food for zooplankton such as
Cladocera, Daphnia sp. and etc. It is suitable
as first larvae feed because its size fits on the
larva’s mouth. Dry weight of Chlorella sp.
contains 38% protein, 5.1% fat, and 24.3%
ash (Fradique et al., 2010). Fish larvae are
the early stage of fish life cycle and
susceptible by pathogen invasion (Puspitasari
et al., 2019). An effort to reduce the risk of
fish larvae died is provide by fed fresh and
lyophilized microalgae (Chlorella,
Scenedesmus, and Haemotococcus), egg
yolk, lyophilized Artemia nuplii (LAN), and
combination of them (Samaee et al., 2021).
Chlorella sp. contains high nutrition and also
be used as a bioremediation agent in reducing
CO2 levels in the air (Cahyonugroho et al.,
2019; Umainana et al., 2019b). Besides,
Chlorella sp. is a coccoid green genus which
one of the most important commercial
microalgae in the world, with the annual
production of biomass exceeds 2,000 tonnes
(Champenois et al., 2015). The ideal growth
specific and the biomass productivity
depends on many factors (Rameshprabu
Ramaraj et al., 2015). These include abiotic,
biotic, and mechanical factors. Abiotic
factors are temperature, minerals, CO2, pH,
water quality, light cycle and intensity; biotic
factors are cell fragility and cell density;
mechanical factors are continuous mixing,
gas bubble size and distribution and mass
transfer, all these are of particular concern in
photo-bioreactors (Ramaraj et al., 2015;
Unpaprom et al., 2015).
Microalgae culture should contain
micronutrients and macronutrients (Şirin &
Sillanpää, 2015). Nutrients classified as
macronutrients are C, H, N, P, K, S, Mg, and
Ca, and micronutrients required by
microalgae are Fe, Cu, Mn, Zn, Co, Mo, Bo,
Vn, and Si (Kawaroe et al., 2019). The
medium that can be used for microalgae
cultivation is the Walne medium. It is often
used as a medium for the culture of
Chlorella sp. It contains Boron (Bo) to
maintain green pigment in Chlorella sp
Jurnal Airaha, Vol.10, No.02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638
(Chilmawati, 2008). In addition, walne also
contains 83.9 mg/l nitrate and 17.7 mg/l
phosphates (Ajijah et al., 2020).
Several researchers have made efforts
to increase nutrients in Chlorella sp. through
various media. They ranged from using
inorganic fertilizers in mass-scale culture to
grow Chlorella sp. (Rahardini et al., 2018), 1
g/L sucrose and 10% inorganic carbon (CO2)
to increase the number of Chlorella sp. (Lin
& Wu, 2015), and adding of 0.5 ml/L walne
fertilizer as a source of microalgae
(Chlorella sp.) nutrition (Purnamawat et al.,
2014). This study aimed to determine the
effect of differences of culture medium
concentration of microalgae Chlorella sp.
METHODS
Time and Location
This study was conducted on January,
2019, at the Natural Feed and Nutrition
Laboratory of the Sorong Marine and
Fisheries Polytechnic.
Procedures
Materials Preparation.
Materials were used in this study was
500 mL Erlenmeyer flask (Pyrex); Dropper
pipette (BRAND) were washed with
detergent and rinsed using flow water then
were dried. Seawater were filtered by filter
paper and sterilization by adding 60 ppm of
chlorine then aerated for 24 hours. Chlorine
on the seawater was neutralized using 20
ppm Na-Thiosulfate. The 200 mL of
seawater were prepared before were added
into 500 mL Erlenmeyer flask and wrapped
using alumunium foil then were prepared for
sterilization to avoid contamination by using
autoclave with temperature of 121oC for at
least 15-20 minutes under 1 psi of preasure.
The seawater were kept at room temperature
(23oC) until use.
Cultivation of Chlorella sp.
The microalgae seeds used in this study
were Chlorella seeds which were harvested
after 7 days and then can be used for
reculture. The seeds was pure Chlorella sp.
which was obtained from Balai Besar
Perikanan Budidaya Air Payau (BBPBAP)
Jepara, West Java. Chlorella sp. was
cultivated on a laboratory scale for seven
days in an Erlenmeyer with sterile seawater.
An inoculum volume was 100 mL were
added into 200 mL of sterile seawater. Walne
dosage was added at the beginning about 2
mL/L of total volume then were kept under
temperature of 23oC, 30 ppt salinity, lighting
for 24 hours, and adequate aeration provided
by blower.
Research methods
This research was an experimental
study with a completely randomized design
(CRD) consisting of four treatments and
three replications. The independent variable
in this study was the difference time in
nutrient concentration adding of the Walne
fertilizer, while the dependent variable was
the population of Chlorella sp. Thus, A
group was added fertilizer only at the
beginning; B group was added fertilizer in
every day; C group was added fertilizer each
two days; and D group was added fertilizer
each three days.
The number of cells was observed
using an electric microscope at 40x
magnification with five fields of view and
three replications for each view. The
observations were made from day 0 (t2) until
day 7 (t1) every 24 hours. The observations
of the number of cells were used to calculate
cell density.
Data Analysis
The observed growth parameters of
Chlorella sp. consisted of peak population,
specific growth rate, and cell density. The
peak population was seen from the highest
cell density value during the culture period.
Microalgae cell density was calculated based
on the formula of (Hutami et al., 2015) as
follows:
1000 x n
N=
LP x p
Note: N : the density of Chlorella sp.
n : the number of observed Chlorella
sp.
LP : field of view
P : the number of field of view
The specific growth rate of microalgae
(µ) was calculated using the formula by (Das
et al., 2011) as follows :
Jurnal Airaha, Vol.10, No.02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638

N2 Cell density of Chlorella sp. showed an

) ln (
µ= N1 increase from the first day to the seventh day
t2-t1 in almost all treatments. Increased cell
Note: N2 : cell density at exponential time density due to Chlorella sp. utilized the
N1 : initial cell density nutrients to diffusion and reproduce. Nutrient
t2 : initial time of cultivation and phytoplankton fraction influenced on
t1 : observation time in exponential growth rates (Juhl & Murrell, 2005). A group
phase showed an increased in cell density
Specific growth rate and density data were continuously from the beginning to the end
analyzed using analysis of variance of the observation. It was suspected that
(ANOVA) Walne fertilizer had given sufficient nutrient
employing Software Minitab version 16. The for cell proliferation. Moreover, other groups
analysis was followed by Tukey’s test with experienced a decrease in cell density on the
(p<0.05) with a 95% confidence level. fifth day (C group) and sixth day (B and D
group). Based on the growth rate phase, it
RESULTS AND DISCUSSION showed differences in each treatment group,
Results but statistically, the treatment group which
The cultivation results of microalgae showed a significant difference was found in
Chlorella sp with different nutrient C group. This proved that C group was the
concentrations are shown in Figure 1. The proper treatment in increasing the production
density cell of Chlorella sp. in each treatment of phytoplankton cells of Chlorella sp.
increased from the beginning of cultivation
to the peak of the highest population and then Table 1. The cell density of Chlorella sp.
decreased. A group showed increasing with varying nutrient concentrations
microalgae cell continuously from the Growth parameter
beginning cultivation to seventh day. B group Treatment µ (day-1) Peak Population
showed increasing in cell density from the (cell/mL)
first day to the sixth day cultivation, then A 0.53 ± 0.08b 2,540,694
decreased in the seventh day. C group B 0.61 ± 0.15b 2,384,996
showed an increasing in cell density from the C 0.83 ± 0.13a 2,628,450
first day to the fifth day, than decreased on
D 0.65 ± 0.08b 2,587,403
the sixth day to seventh day. D group
experienced an increase in the number of cell
density from the first day to the sixth day and Based on ANOVA, Chlorella sp. showed a
decreased on the seventh day. different significance (p<0.05) was the C
3,000,000 group with the highest specific growth rate
than other groups. The highest cell density
2,500,000 (peak population) of C group was 2,628,450
cell/mL with an average 1,814,175 cell/mL
density (cell/ml)

2,000,000
(Fig.1). Moreover, another group showed no
1,500,000 A
significant difference with the lowest specific
B
1,000,000 growth rate beingC A group, but the lowest
500,000 cell density was D B group with value
2,384,996 cells/mL, in an average cell
- density was 1,501,365 cells/mL.
0 1 2 3 4 5 6 7
Culture period (days) Discussion
The nutrient addition of walne
Figure 1. Growth rate phase of Chlorella sp. fertilizer every two days could increase the
with varying nutrient concentrations cell density of Chlorella sp. The nutrients
Jurnal Airaha, Vol.10, No.02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638
needed by phytoplankton were well utilized
and reached an exponential phase
significantly, compared to the other treatment
groups. Generally, microalgae growth is
divided into 4 (four) phases which are lag
phase, log or exponential phase, stationary
phase and death phase (Moazami et al.,
2012). The availability of nutrients in a
microalgae growth medium affects the
growth rate. Futhermore, (Sánchez et al.,
2000) stated that the culture medium greatly
affects the biomass yield and protein content
of microalgae (Chlorella). Based on R.
Ramaraj et al., (2016) and Rameshprabu
Ramaraj et al., (2015), explained that algae
are easy to grow and cultivate anywhere with
less energy requirements and few nutrients.
However, the addition of nutrients to
Chlorella culture media made a significant
contribution to growth and could even
increase biomass productivity and nutritional
value (Kim et al., 2013; Blair et al., 2014). B
group showed differences in the level of cell
density compared to the C group, presumably
due to the nutrients were given was excesive
that Chlorella sp unable to absorb nutrients
effectively, leading to the buildup of toxic
organic matter that decreased growth
eventually. Excessive nutrients will be toxic
to microalgae growth, causing growth to be
suboptimal (Arinta, 2012; Swandewi et al.,
2017). The specific growth rate can also be
used as an indicator of the carrying capacity
of the medium and the availability of energy
for cells to divide (Wahyuni et al., 2019;
Musa et al., 2013). The low growth rate may
be caused by nitrate and phosphate, which
cells cannot adequately utilize for growth and
division. According to Arinti, (2012) and
Swandewi et al., (2017), excess phosphate
concentration will inhibit the process of
assimilation of phosphorus, while low
phosphate concentration will inhibit the
formation of Adenosine Tri Phosphate (ATP)
for cell growth. Compound N in NH 4+
assimilates with glutamic acid into
macromolecules needed by microalgae cells.
According to Blair et al., (2014), the
concentration of N and P by 50-100% is
optimal for algae growth.
The addition of Walne media every
day can produce the number of particles in
the culture media. The greater the number of
particles in the culture medium, the longer
the time required to pass through the
adaptation phase. These particles can
increase the value of turbidity, BOD and
COD so that photosynthesis becomes more
inhibited and also slows down the growth
rate of Chlorella sp. (Dianursanti et al.,
2014). Then the D group showed a lower
level of cell density as well, this is
presumably due to the nutrients that can be
used by Chlorella sp. enough but not
optimal. Nutrient composition plays an
important role in microalgae cultivation,
excessive or depletion source of nutrient
might affect the biomass quality (Yaakob et
al., 2021). Dianursanti et al., (2014) stated
that Chlorella biomass growth using Walne
medium was more stable than using liquid
waste from tofu because Walne media
contained micro nutrients such as Fe, Mn,
Mg and Cl.
Conclusion
Statistically, the addition of culture
medium every two days (C group) showed a
significant difference (P<0.05) on the
specific growth rate of microalgae Chlorella
sp. with a value of 0.83 ± 0.13 µ (day -1).
However, the difference in nutrient
concentration does not have a different effect
on the cell density of Chlorella sp. The
highest population peak was found in
treatment C, with a value of 2,628,450
cells/ml. There is a need for mass culture
assay to determine the level of effectiveness
and efficiency.
Jurnal Airaha, Vol.10, No.02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638

References C., Gouveia, L., Bandarra, N. M., &

Ajijah, N., Tjandra, B. C., Hamidah, U., Raymundo, A. (2010). Incorporation of
Widyarani, & Sintawardani, N. (2020). Chlorella vulgaris and Spirulina maxima
Utilization of tofu wastewater as a biomass in pasta products. Part 1:
cultivation medium for Chlorella Preparation and evaluation. Journal of
vulgaris and Arthrospira platensis. IOP the Science of Food and Agriculture,
Conference Series: Earth and 90(10), 1656–1664.
Environmental Science, 483(1). https://doi.org/10.1002/jsfa.3999
https://doi.org/10.1088/1755- Hutami, F. E., Supriharyono, & Haeruddin.
1315/483/1/012027 (2015). Laju Filtrasi Kerang Hijau
Arinti, R. (2012). Effect Concentration of (Perna viridis) Terhadap Skeletonema
Liquid Waste Tapioca Growth and costatum Pada Berbagai Tingkat
Lipid Levels Generated By Salinitas. Ejournal, 4, 125–130.
Scenedesmus sp. State Juhl, A. R., & Murrell, M. C. (2005).
IslamicUniversity (UIN) Maulana Malik Interactions between nutrients,
Ibrahim Malang. phytoplankton growth, and
Blair, M. F., Kokabian, B., & Gude, V. G. microzooplankton grazing in a Gulf of
(2014). Light and growth medium effect Mexico estuary. Aquatic Microbial
on Chlorella vulgaris biomass Ecology, 38(2), 147–156.
production. Journal of Environmental https://doi.org/10.3354/ame038147
Chemical Engineering, 2(1), 665–674. Kawaroe, M., Prartono, T., Sunuddin, A., &
https://doi.org/10.1016/j.jece.2013.11.0 Sari, D. (2019). Mikroalga potensi dan
05 pemanfaatannya untuk produksi bio
Champenois, J., Marfaing, H., & Pierre, R. bahan bakar.
(2015). Review of the taxonomic https://books.google.com/books?
revision of Chlorella and consequences hl=id&lr=&id=AI8SEAAAQBAJ&oi=f
for its food uses in Europe. Journal of nd&pg=PP1&dq=Kawaroe,+M.,
Applied Phycology, 27(5), 1845–1851. +Prartono,+T.,+Sunuddin,+A.,+Sari,
https://doi.org/10.1007/s10811-014- +D.+W.,+%26+Augustine,+D.+
0431-2 %09(2019).+Mikroalga+
Chilmawati, D. (2008). Penggunaan Media %09potensi+dan+pemanfaatannya+untu
Kultur Yang Berbeda Terhadap k+produksi+bio+%09bahan+bakar.
Pertumbuhan Chlorella sp The Used of +PT+Penerbit+IPB+Press.+
Different Culture Medium on the Kim, J., Yoo, G., Lee, H., Lim, J., Kim, K.,
Growth of Chlorella sp. 4(1), 42–49. Kim, C. W., Park, M. S., & Yang, J. W.
Das, P., Lei, W., Aziz, S. S., & Obbard, J. P. (2013). Methods of downstream
(2011). Enhanced algae growth in both processing for the production of
phototrophic and mixotrophic culture biodiesel from microalgae.
under blue light. Bioresource Biotechnology Advances, 31(6), 862–
Technology, 102(4), 3883–3887. 876.
https://doi.org/10.1016/j.biortech.2010.1 https://doi.org/10.1016/j.biotechadv.201
1.102 3.04.006
Dianursanti, Rizkytata, B. T., Gumelar, M. Lin, T. S., & Wu, J. Y. (2015). Effect of
T., & Abdullah, T. H. (2014). Industrial carbon sources on growth and lipid
tofu wastewater as a cultivation medium accumulation of newly isolated
of microalgae Chlorella vulgaris. microalgae cultured under mixotrophic
Energy Procedia, 47, 56–61. condition. Bioresource Technology,
https://doi.org/10.1016/j.egypro.2014.01 184, 100–107.
.196 https://doi.org/10.1016/j.biortech.2014.1
Fradique, Ḿonica, Batista, A. P., Nunes, M. 1.005
Jurnal Airaha, Vol.10, No.02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638

Moazami, N., Ashori, A., Ranjbar, R., Sustainable and Green Energy. Special
Tangestani, M., Eghtesadi, R., & Nejad, Issue: Renewable Energy Applications
A. S. (2012). Large-scale biodiesel in the Agricultural Field and Natural
production using microalgae biomass of Resource Technology, 4(1), 13–19.
Nannochloropsis. Biomass and https://doi.org/10.11648/j.ijrse.s.201504
Bioenergy, 39, 449–453. 0101.13
https://doi.org/10.1016/j.biombioe.2012. Samaee, S. M., Atashbar Kangarloei, B.,
01.046 Noori, F., & Estévez, A. (2021). An
Mulyanto, A. (2010). Penambat Gas Karbon Endeavor to Find Starter Feed
Dioksida. J. Hidrosfir Indonesia, 5(2), Alternatives and Techniques for
13–23. Zebrafish First-Feeding Larvae: The
Musa, B., Raya, I., Makassar, S. D.-, Effects on Viability, Morphometric
Hasanuddin, U., & 2013, U. (2013). Traits, Digestive Enzymes, and
Pengaruh penambahan ion Cu2+ Expression of Growth-Related Genes.
terhadap laju pertumbuhan fitoplankton Zebrafish, 18(1), 73–91.
Chlorella vulgaris. Hasanuddin https://doi.org/10.1089/zeb.2020.1928X
University Repository, 1–9. Sánchez, S., Martínez, M. E., &
https://core.ac.uk/download/pdf/254939 Espinola, F. (2000). Biomass production
00.pdf and biochemical variability of the marne
Purnamawat, F. S., Soeprobowati, T. R., & microalga Isochrysis galbana in relation
Izzati, M. (2014). Potensi Chlorella to culture medium. Biochemical
vulgaris Beijerinck Dalam Remediasi Engineering Journal, 6(1), 13–18.
Logam Berat Cd Dan Pb Skala https://doi.org/10.1016/S1369-
Laboratorium. Bioma : Berkala Ilmiah 703X(00)00071-1
Biologi, 16(2), 102. Swandewi, I. G. A. P. A. P., Anggraeni, A.
https://doi.org/10.14710/bioma.16.2.102 A. M. D., & Admadi, B. (2017).
-113 Pengaruh Penambahan NaNO3 Dan
Puspitasari, A. W., Arfiati, D., & Hu, S. K2HPO4 Pada Media BG-11 Terhadap
(2019). Can Cinnamaldehyde Increase Konsentrasi Biomassa Dan Klorofil
The Innate Immune System on Medaka Tetraselmis chuii. Jurnal Rekayasa Dan
Larvae ? 2(1), 21–26. Manajemen Agroindustri, 5(1), 1–11.
https://doi.org/10.11594/bbrj.02.01.04
Rahardini, R. A., Helmiati, S., & Triyatmo,
B. (2018). Effect of inorganic fertilizer Şirin, S., & Sillanpää, M. (2015). Cultivating
on the growth of freshwater Chlorella and harvesting of marine alga
sp. IOP Conference Series: Earth and Nannochloropsis oculata in local
Environmental Science, 139(1). municipal wastewater for biodiesel.
https://doi.org/10.1088/1755- Bioresource Technology, 191, 79–87.
1315/139/1/012005 https://doi.org/10.1016/j.biortech.2015.0
Ramaraj, R., Unpaprom, Y., & Dussadee, N. 4.094
(2016). Cultivation of Green Microalga, Umainana, M. R., Mubarak, A. S., &
Chlorella vulgaris for Biogas Masithah, E. D. (2019b). Pengaruh
Purification. International Journal of Konsentrasi Pupuk Daun Turi Putih
New Technology and Research, 2(3), (Sesbania grandiflora) Terhadap
263569. Populasi Chlorella sp. Journal of
Ramaraj, Rameshprabu, Dussadee, N., Aquaculture and Fish Health, 8 (1), 1.
Whangchai, N., & Unpaprom, Y. https://doi.org/10.20473/jafh.v8i1.11219
(2015). Microalgae Biomass as an Unpaprom, Y., Tipnee, S., & Rameshprabu,
Alternative Substrate in Biogas R. (2015). Biodiesel from green alga
Production. International Journal of Scenedesmus acuminatus. International
Jurnal Airaha, Vol.10, No.02 (Dec 2021):295 – 294, p-ISSN 2301-7163, e-ISSN 2621-9638

Journal of Sustainable and Green

Energy, 4(1), 1–6.
https://doi.org/10.11648/j.ijrse.s.201504
0101.11
Wahyuni, N., Setya Rahardja, B.,
Muhammad, D., Azhar, H., Akuakultur,
J., Perikanan, F., Kelautan, D.,
Airlangga Di Banyuwangi, U., &
Airlangga, U. (2019). The Effect of
Giving Combination Concentration of
Leaves of Moringa oleifera with Walne
Fertilizer in Culture Media on the
Growth and Content of Carotenoids in
Dunaliella salina. Journal of
Aquaculture Science, 4(1), 37–49.
https://doi.org/10.31093/joas.v4i1.67
Yaakob, M. A., Mohamed, R. M. S. R., Al-
Gheethi, A., Ravishankar, G. A., &
Ambati, R. R. (2021). Influence of
nitrogen and phosphorus on microalgal
growth, biomass, lipid, and fatty acid
production: An overview. Cells, 10(2),
1–19.
https://doi.org/10.3390/cells10020393