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ISSN 0854-5154 | eISSN 2442-7349
2020
Optimizing Photoperiodism, Growth Media Dilution, and Frequency of
Inoculum Addition in Microalgae (Chlorella vulgaris) Culture Using Bi-
oslurry (ADDMW) as Growth Media
T. Taufikurahman1 and Teguh Adhitia Suyadi1,*,
1Sekolah Ilmu dan Teknologi Hayati, Institut Teknologi Bandung.
*Corresponding author. Email: teguhadhitiasuyadi7@gmail.com
ABSTRACT
In biogas production using dairy manure, an anaerobic digestion process also produced a by product bioslurry, also known as Anaer-
obically Digested Dairy Manure Wastewater (ADDMW), which is rich in ammonium and ortophosphate. ADDMW is potentially to
be used as growth media to grow autotrophic organism, including microalgae. Furthermore, microalgae potentially could reduce
organic content in ADDMW, thus play a role as phycoremediator to organic wastewater. In this study we used ADDMW medium for
the growth of microalgae, determining its growth kinetics and level of reduction in ammonium and orthophosphate content in
ADDMW. In addition, we also analysed protein content in microalgae biomass for potential use as animal feed. The experiment was
conducted using some variations in photoperiodism, i.e., 16:8; 12:12; and 8:16 and medium dilution factor of 2.5 and 5 times. Fur-
thermore, frequency of inoculum addition was also examined i.e., a) 80 mL on day 0 (once); b) 26.67 mL on day 0 to 2 (first 3 days);
and c) 8 mL on days 0 to 9 (every day). The results showed that 16:8 photoperiod and 5 times medium dilution produced the highest
biomass growth and productivity kinetics. Variation in frequency of inoculum addition showed no significant effect to a decrease in
ammonium levels but indicated some effect to orthophosphate level. It can be concluded that microalgae can be grown in ADDMW
media and can reduce its ammonium and ortophosphate content, as a phycoremediator, before the wastewater is dumped to the river.
Keywords: bioslurry, ADDMW, microalgae, Chlorella vulgaris, phycoremediation © Institut Teknologi Bandung. All rights reserved.
Received 18 Juni 2019 • Revisied 24 Juni 2019 • Accepted 2 Juli 2019 • Available online 16 September 2020
INTRODUCTION
Livestock farming produces large amounts of waste. The
waste comes from the metabolic process of cows (faeces and
urine) and remaining feeds. Farmers who do not have suffi-
cient waste treatment technologies and has little concern on
environmental risk would dispose the waste directly to open
stream. These activities certainly have a negative impact on
the environment, such as the occurrence of eutrophication due
to the uncontrolled supply of nutrients (ammonium and phos-
phate), increased biological oxygen demand (BOD), changes
in odor and reduced the clarity of water. Increasing the de-
mand for beef and cow's milk from the consumens certainly
requires a good waste treatment system to maintain a good
environmental condition.
The use of cattle dairy waste as a substrate in biogas produc-
tion is an option that can be chosen by farmers. From anaero-
bic process of biogas production, bioslurry will be released as
a by product. Bioslurry contains ammonium and total phos-
phate (TP) with concentrations reaching 136 and 34 mg/L re-
spectively [1]. For companies that process livestock breeding
waste in raw materials for the production of large quantities
of biogas, bioslurry is also another problem because it is pro-
duced continuously in large quantities. The nutrients con-
tained in the bioslurry, such as ammonium and phosphate, can
be used as a source of nourishment in the microalgae growing
medium.
The cultivation of microalgae generally uses synthetic me-
dium that certainly requires costs in its supply, which will
therefore influence the selling price of products processed by
microalgae. The cultivation of microalgae using a waste me-
dium will provide two advantages at the same time, reduced
cultivation costs and environmentally friendly waste treat-
ment.
Microalgae Chlorella vulgaris has been cultivated widely
since it has been known to have a lot of benefits, especially its
high protein content. Chlorella vulgaris naturally grows in
open water with a source of nutrients derived from water.
Chlorella vulgaris plays an important role in the food chain of
the aquatic system as a source of nourishment for other organ-
isms [2]. The high protein content in microalgae biomass
makes it feasible to use it as an alternative mixture of feed
production, considering that the intake of protein in animal
Please cite this article as: Taufikurahman, T., Suyadi, T.A., Optimizing Photoperiodism, Growth Media Dilution, and Fre-
quency of Inoculum Addition in Microalgae (Chlorella vulgaris) Culture Using Bi-oslurry (ADDMW) as Growth Media, J.
Matem. & Sains, 2020, 25, 6-10.
DOI Number: 10.5614/jms.2020.25.1.2
7
© Faculty of Mathematics and Natural Sciences ISSN 0854-5154 | eISSN 2442-7349
Institut Teknologi Bandung. All rights reserved.
Taufikurahman et al. / Jurnal Matematika & Sains, 25, 2020, 6-10
feed currently adheres to soy flour and fish meal that compete
directly in its uses as ingredients for human consumption. Fur-
thermore, Ursu et al. [3] states that there are 18 amino acids
present in microalgae proteins. Of these 18 amino acids, 9 of
them are essential amino acids for humans.
EXPERIMENTAL
Determination of Photoperiodism and Dillution Factor
Bioslurry is a liquid residue from the anaerobic process of cat-
tle farm waste. The bioslurry comes from Animal Husbandry
Faculty, Padjajaran University. Bioslurry still contains vari-
ous solids so it must go through the filtering stage before using
it. Bioslurry filtrate produced by filtration using cotton was
diluted using 2.5 and 5 times using distilled water. Bioslurry
is not through the sterilization process first. This is intended
to observe whether microalgae growth will be hampered or
not.
A total of 720 ml of diluted medium was mixed with 80 ml of
inoculum which contain 107 cells/ml. The cultivation was car-
ried out in a photobioreactor with lighting derived from a 100
μmol photon m-2s-1 fluorescent lamp (Philips Cool White,
TLD 36 Watt). The culture was kept at a temperature of 27-
30°C and pH 7-8. Aeration was given at a rate of 1 L/minute.
Photoperiodization was given 16:8, 12:12 and 8:16. Photo-
periodization variations are intended to see the dark and bright
regimes that are most suitable to be applied to the system. The
cultivation was carried out for 10 days period.
The experimental design for determining photoperiodization
and the mean dilution factor of ADDMW can be seen in Table
1.
Tabel 1. Photoperiodism and Dillution Factor Variation
Photoperiodism
Dillution Factor
16:8
2.1. 2.5
2.2. 5
12:12
8:16
Frequency of Inoculum Addition
The addition of the inoculum is varied as shown in Table 2.
The inoculum is set to be in a similar condition every time the
addition takes place. In Sample A, the inoculum was added
directly on D-0. In sample b, the inoculum was added in the
first 3 days of cultivation. in sample c, the inoculum is added
every day during cultivation. The experimental design for
determining variation in frequency of the inoculum addition
can be seen in Table 2.
Each sample was tested in 3 repetitions. Control 1 is a sample
with a synthetic medium (Bold Basal Medium). Control 1 was
used to compare the different of the growth of microalgae
cultivated in waste-based medium and synthetic medium.
Control 2 is a bioslurry medium without inoculum. Control 2
was used to compare the ammonium and ortophosphate
profile during cultivation with Sample A.
Specific growth rate
Measurement of microalgae growth begins with making a
microalgae dry weight curve against the absorbance of
microalgae. The microalgae wet sample is serially diluted and
then dried to a constant weight. Wet samples are measured
first for absorbance. The growth of microalgae was analyzed
using wavelength absorption data using a spectrophotometer.
The wavelength used was 680 nm. Absorbance measurements
were performed every day, starting from day 0 to day 10.
Specific growth rates are defined as biomass growth rates per
unit of time. Specific growth rate of microalgae specified
during exponential phase cultures were based on a growth
curve obtained. The specific growth rates (µ) were determined
using the following equation [4]:
--------------------------------------------------------------------------------
ABSTRAK: Dalam produksi biogas dengan menggunakan kotoran
sapi, proses penguraian anaerob juga menghasilkan produk samping
bioslurry
yang disebut juga dengan
Anaerobically Digested Dairy Ma-
nure Wastewater
(ADDMW) yang kaya akan ammonium dan or-
tophosphate. ADDMW berpotensi digunakan sebagai media pertum-
buhan untuk menumbuhkan organisme autotrof, termasuk mikroalga.
Selain itu mikroalga berpotensi dapat menurunkan kandungan organik
pada ADDMW, sehingga berperan sebagai fikoremediator air limbah
organik. Pada penelitian ini digunakan media ADDMW untuk per-
tumbuhan mikroalga, serta dilakukan penentuan kinetika pertum-
buhannya dan tingkat penurunan kandungan amonium dan ortofosfat
dalam ADDMW. Selain itu, kami juga menganalisis kandungan pro-
tein dalam biomassa mikroalga untuk potensi penggunaan sebagai pa-
kan ternak. Percobaan dilakukan dengan menggunakan beberapa vari-
asi fotoperiodisme, yaitu 16:8; 12:12; dan 8:16 serta faktor pengenc-
eran medium 2,5 dan 5 kali. Selanjutnya frekuensi penambahan inoku-
lum juga dieksaminasi yaitu a) 80 mL pada hari ke 0 (satu kali); b)
26,67 mL pada hari 0 sampai 2 (3 hari pertama); dan c) 8 mL pada
hari 0 sampai 9 (setiap hari). Hasil penelitian menunjukkan bahwa
penyinaran 16:8 dan pengenceran medium 5 kali menghasilkan per-
tumbuhan biomassa dan kinetika produktivitas tertinggi. Variasi frek-
uensi penambahan inokulum tidak menunjukkan pengaruh yang sig-
nifikan terhadap penurunan kadar amonium, tetapi menunjukkan
adanya pengaruh terhadap kadar ortofosfat. Dapat disimpulkan bahwa
mikroalga dapat dibudidayakan pada media ADDMW dan dapat
menurunkan kandungan amonium dan ortofosfatnya sebagai fikore-
mediator sebelum limbah cair dibuang ke sungai.
Kata kunci:
bioslurry
, ADDMW, mikroalga,
Chlorella vulgaris
, fikore-
mediasi
__________________________________________________
8
© Faculty of Mathematics and Natural Sciences ISSN 0854-5154 | eISSN 2442-7349
Institut Teknologi Bandung. All rights reserved.
Taufikurahman et al. / Jurnal Matematika & Sains, 25, 2020, 6-10
Table 2. Inoculum Adding Frequency Variation
Sample
Inoculum Addition (mL/day)
D-0
D-1
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
A
80
-
-
-
-
-
-
-
-
-
B
26,7
26,7
26,7
-
-
-
-
-
-
-
C
8
8
8
8
8
8
8
8
8
8
Control 1
80
-
-
-
-
-
-
-
-
-
Control 2
-
-
-
-
-
-
-
-
-
-
where x is the dry weight of biomass at the end of the expo-
nential phase (g/L), x0 is the dry weight of the biomass at the
beginning of the exponential phase (g/L) and t is the temporal
duration of the exponential phase (days). The doubling time
is the time required by the organism to multiply the amount of
biomass (biomass at the moment doubled by initial biomass).
The multiplication time (dt) can be determined using the fol-
lowing equation:
with dt is a multiplication time, and μ is a specific growth rate
of microalgae (gram/day).
Ammonium Content
The ammonium content test was performed to experiments
with variations in the Table 2, using the Nessler method. The
sample was centrifugated to obtain the supernatant. Centrifu-
gation was performed on D-0, D-3. D-5, D-7, and D-10. Cen-
trifugation was performed at a rate of 4500 g for 5 minutes. A
total of 10 mL of supernatant was added with 0.2 mL of ness-
ler reagent then stirred using vortex. After 10 minutes without
any treatment, the sample absorbance was measured using a
spectrophotometer at a wavelength of 420 nm. The absorb-
ance value obtained was converted into ammonia concentra-
tion using the equation of the standard curve that has been
carried out. Process on making standard curve was absolutely
the same with the process on testing the sample, but the sam-
ple was replaced with ammonium nitrate.
Orthophosphate Content
Orthophosphate content was measured in an experiment with
variations in the frequency of inoculum addition. The reagents
used for the test were ammonium molybdate and SnCl2. The
sample was centrifugated at a rate of 4500 g for 5 minutes to
obtain the supernatant. A total of 25 ml of the test sample was
put in erlenmeyer and then added with 1 mL of ammonium
molybdate reagent while stirred slowly. After 20 minutes, 2
drops of SnCl2 solution were added to the sample. The absorb-
ance of the samples was tested using a spectrophotometer at a
wavelength of 650 nm. The absorbance value was then con-
verted into orthophosphate concentration using the equations
obtained from the standard orthophosphate curve. The standar
curve was made with the same process on testing the sampel,
but the sampel was replaced with phosphoric acid.
RESULTS AND DISCUSSION
Growth Rate Kinetic
The variations of photoperiodization significantly influenced
the amount of dry biomass obtained at the end of the exponen-
tial phase (P<0.05). The dry biomass at the end of the expo-
nential phase for the 16:8 photoperiodization treatment in
each medium was the highest compared to other variations. In
ADDMW medium with dilution 2.5 times, dry biomass was
obtained at the end of the log phase was 0.945 ± 0.034 g/L,
while 12:12 photoperiodization was 0.678 ± 0.023 g / L and
8:16 photoperiodization was 0.663 ± 0.032 g / L. The same
trend also occurred for the treatment of 5 times dilution. 16: 8
photoperiodization was 0.893 ± 0.056 g/L, photoperiodization
of 12:12 was 0.789 ± 0.050 g/L and photoperiodization of
8:16 was 0.779 ± 0.030 g /L. The specific growth rate and the
time of multiplication of the biomass in experiments with pho-
toperiodization and dilution factors are shown in Table 3.
The multiplication time of the biomass at 16: 8 photoperiodi-
zation occurs more rapidly in 2.5 or 5 times the average dilu-
tion (Table 3). The specific growth rate is an exact number
that can be used to determine that an organism is able to adapt
to a certain culture condition [5]. Photoperiodization is one of
the factors that influence the physiology of the plant.
The 16:8 photoperiodization treatment produced the highest
amount of biomass at the end of the log phase, the specific
growth rate, the multiplication time and the biomass produc-
tivity compared to other variations (Table 3). Photoperiodiza-
tion will directly influence the photosynthesis system carried
out by microalgae. Correct photoperiodization will produce
an optimal production of ATP and NADH, with that energy
which will then be used in the dark reactions for the growth
of microalgae [6]. 16:8 photoperiodization will produce a
more optimal process of photosynthesis than other variations.
16 hours when irradiation is given, it will be used by the mi-
croalgae chlorophyll to convert light energy into ATP and
NADPH. The ATP and NADPH products are used to convert
CO2 into glyceraldehyde-3-phosphate (G3P). This part of
G3P will be used for the formation of carbohydrates, such as
glucose and starch. Another part of G3P will be used for the
formation of pyruvate, which will then be converted into ace-
tyl co-A through the glycolysis reaction. The resulting acetyl
co-A will be converted to malonyl co-A. Malonyl co-A is the
initial form of lipids accumulated in microalgae [7].
9
© Faculty of Mathematics and Natural Sciences ISSN 0854-5154 | eISSN 2442-7349
Institut Teknologi Bandung. All rights reserved.
Taufikurahman et al. / Jurnal Matematika & Sains, 25, 2020, 6-10
Table 3. Specific Rate and Time of Multiplication of Microalgae Chlorella vulgaris Biomass with Frequency Variations of
Inoculum Addition and Medium Dilution (each variation was tested on 3 repetition).
Statistical tests showed that the average dilution had a signif-
icant effect on the multiplication time of the biomass
(P<0.05). Chen et al. [8] stated that the lowest specific growth
rate at 2.5 times dilution was caused by the increased of tur-
bidity of the medium due to more content of colloid and or-
ganic material in it. A higher level of turbidity will reduce the
penetration of light into culture so that photosynthesis can be
inhibited. However, the dilution factor applied to the experi-
ment with the frequency variation of the addition of a fixed
inoculum is 2.5 times. This is done to confirm that the purpose
of the research is to remedy the waste. Eventually, a percent-
age reduction of ammonium and orthophosphate levels in 2.5
times diluted waste will be assessed using the phycoremedia-
tion method.
Ammonium Content
The ammonium content in the medium will be directly related
to the accumulation of metabolites in microorganisms. The
accumulation of biomass occurs higher (if compared to the li-
pid accumulation) when the soil has a sufficient nitrogen and
phosphate content or in other words does not show nitrogen
and phosphate deficiency [9]. The ammonium content was an-
alyzed on ADDMW medium of samples with different fre-
quencies of inoculum addition. The profile of changes in the
ammonium content in the ADDMW medium is shown in Ta-
ble 5.
Table 5. Ammonium Reduction Percentage
Initial concentration of ammonium was measured for sample
on D-0 of cultivation. Final concentration was measured on
D-10 of cultivation. Microalgae use ammonium for the supply
of Nitrogen which will then be used to carry out some prsoes
metabolism, such as protein formation and energy formation.
Statistical tests have shown that the frequency of addition of
inoculum did not significantly influence the reduction of am-
monium levels in the medium. In each treatment a reduction
of ammonium levels of more than 95% was found. In the pre-
vious research by Wang et al. [10], a reduction of 93.6% am-
monium levels in wastewater manure that went through the
stages of anaerobic bacteria digesting using remediator Chlo-
rella vulgaris agent. Nitrification bacteria present in the me-
dium to convert the ammonium content into nitrate [11]. nitri-
fying bacteria that live in heterotrophic aerobics and can grow
well in the growing system for growing conditions are very
suitable for supporting the growth of bacteria (aeration and
ammonium availability as a nitrifying substrate). This is evi-
denced by the presence of high nitrate levels due to the nitri-
fication process in the ADDMW medium as originally almost
no increase of about 5 ppm at the end of cultivation (data ob-
tained from the control treatment test, in the form of ADDMW
medium by aeration and exposure light without the addition
of inoculum). Furthermore, the ammonium in the medium is
also used by microalgae to grow. The presence of nitrifying
bacteria has an impact on the reduction of the ADDMW am-
monium content. Therefore, a reduction of the ammonium
profile levels over a whole variety of relatively different treat-
ment, which means that the reduction of ammonium in the soil
remains the case if it is caused by consumption of microalgae
or bacterial nitrification.
Orthophosphate Content
Phosphate is one of the macronutrient elements necessary for
plants growth. Phosphate will be used by microalgae for the
Specific Growth Rate
(day-1)
Multiplication Time
(day)
Biomass Productivity
(g/L-1day-1)
16:8
12:12
8:16
16:8
12:12
8:16
16:8
12:12
8:16
ADDMW
0.276
0.230
0.266
2.517
3.030
2.625
0,109
0,104
0,105
2.5
±
±
±
±
±
±
±
±
±
0,017
0.021
0.027
0.160
0.290
0.247
0.025
0.005
0.006
ADDMW 5
0.385
±
0.018
0.342
±
0.010
0.258
±
0.013
1.799
±
0.086
2.028
±
0.063
2.682
±
0.138
0.118
±
0.007
0.117
±
0.003
0.101
±
0.001
Inoculum Adding
Frequency
Initial Concentration
(mg/L)
Final
Concentration (mg/L)
Reduction
Percentage (%)
Sample A
123.992
5.349
95.69 ± 0.767
Sample B
141.799
5.915
95.82 ± 0.124
Sample C
141.798
4.117
97.10 ± 0.135
Control 2
147.835
6.204
95.80 ± 1.462
10
© Faculty of Mathematics and Natural Sciences ISSN 0854-5154 | eISSN 2442-7349
Institut Teknologi Bandung. All rights reserved.
Taufikurahman et al. / Jurnal Matematika & Sains, 25, 2020, 6-10
synthesis of phospholipids, nucleic acids and cell division [12,
13]. The orthophosphate content in the ADDMW medium is shown in Table 6. The frequency of inoculum addition signif-
icantly influenced the decrease in orthophosphate levels in the
ADDMW medium.
Table 6. Orthophosphate Reduction Percentage
Inoculum Adding
Frequency
Initial Concentration
(mg/L)
Final Concentration
(mg/L)
Reduction Percent-
age (%)
Sample A
7.451
1.598
78.55 ± 1.788
Sample B
6.151
1.824
70.35 ± 2.800
Sample C
5.768
2.001
65.30 ± 2.373
Control 2
7.451
1.598
78.55 ± 1.788
CONCLUSION
Photoperiodism of 16:8 with a dilution factor of 2.5 times
made a positive effect on the biomass of microalgae, with the
highest dry biomass of 0.945 ± 0.034 g/L. Completting total
inoculation at the beginning of cultivation produced highest
specific growth rate and quickest doubling time compared to
other treatments. Variation in the frequency of inoculum ad-
dition did not affect ammonia levels of bioslurry. However,
reduction percentage in orthophosphate levels showed signif-
icant different with various frequency of inoculum addition,
i.e 78.55 ± 1.787 (once), 70.35 ± 2.800 (first 3 days), and
65.30 ± 2.373 (daily).
ACKNOWLEDGEMENT
The author would like to thank Lembaga Penelitian dan
Pengabdian Masyar ITB (LPPM ITB) for providing funding
for this research through the P3MI program in 2018.
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