Materials Today: Proceedings 87 (2023) 263–271

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Rotary Algae Biofilm Reactor (RABR) using microalgae Chlorella sp. for
tofu wastewater treatment
Shinta Elystia ⇑, Fakhriyah Hanifa Mazaya Nasution, Aryo Sasmita
Environmental Engineering Program, Chemical Engineering Department, Riau University, Pekanbaru 28293, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history: Indonesia was known to be dominated by the household-scale tofu industry, with limited funds to treat
Available online 27 March 2023 the tofu wastewater produced. Therefore, the producers choose to directly discharge the tofu wastewater
to a ditch or other water body without appropriate treatment. Tofu wastewater can pollute the aquatic
Keywords: environment due to its content, consisting of organic compounds and high nutrients. Utilization of tofu
Chlorella sp. wastewater as a source of nutrients to support the growth of microorganisms such as the microalgae
Rotary Algae Biofilm Reactor (RABR) (Chlorella sp.) can be an alternative to the problem of tofu wastewater using Rotary Algae Biofilm
Tofu wastewater
Reactor (RABR) technology. This study aims to determine the growth rate of microalgae and the removal
of COD and NH3 parameters by cultivating Chlorella sp. On a medium with various treatments of tofu
wastewater concentration, namely 40%, 60%, 80%, and 100%, and contact time within (0, 1, 3, and 5 days).
The results showed that in the treatment of 40% tofu wastewater concentration on day 5, the highest COD
and NH3 removal efficiencies were 75.88% and 80.45%, respectively, with the total density of suspended
and attached cells of Chlorella sp. of 3.99
106 cells/mL.
Ó 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (https://creativecommons.org/licenses/by-nc-nd/4.0).
Selection and peer-review under responsibility of the scientific committee of the 3rd International Con-
ference on Chemical Engineering and Applied Sciences.

1. Introduction BOD of 6,000–8,000 mg/l, and ammonia levels of 23.3–23.5 mg/l

Tofu is one of the local food ingredients that has become chiefly
all levels of society’s favorite in Indonesia because it provides good
nutrition, is affordable, and has a savory taste. The development of
the tofu industry in Indonesia was mainly dominated by small and
medium-scale businesses with limited funds to process the result-
ing liquid waste [1]. As a result, mostly the tofu producers directly
dispose of the liquid waste into surface water without prior treat-
ment, causing environmental problems [2]. It is known that the
amount of liquid waste produced by the tofu industry is quite
large; the processing of each kilogram of soybean can produce as
much as 43.5 L of tofu wastewater [3].
The tofu wastewater produced was mostly a thick liquid that is
separated from the tofu clumping process or referred to as ‘‘whey”
[4]. The high content of organic substances such as protein, carbo-
hydrates, and fat causes high levels of organic substances and
nutrients in tofu wastewater [5]. According to Faisal [6], tofu
wastewater has COD levels ranging from 7,500–14,000 mg/l, a
⇑ Corresponding author.
E-mail address: shinta.elystia@lecturer.unri.ac.id (S. Elystia).
with a pH of 5–6. With the content of substances that have passed
quality standards, if tofu wastewaters are disposed into bodies of
water continuously, it will decrease water quality, especially the
emergence of foul odors and disrupt aquatic ecosystems [3].
Tofu wastewater treatment is known to be carried out biologi-
cally by utilizing the activity of microorganisms that can decom-
pose pollutants in the waste. Aerobic biological treatment
technologies that are often used include stabilization ponds, trick-
ling filters, and activated sludge processes, which are still conven-
tional, require a large area of land, have high energy consumption,
have a costly operating process, and produce excess sludge [7].
Therefore, microalgae-based wastewater treatment is known to
be an alternative to conventional wastewater treatment and a sus-
tainable and cost-effective technology due to the ability of microal-
gae to have a high absorption capacity of nutrients and organic
substances and their great potential in the CO2 fixation process
through photosynthesis.
Chlorella sp. Microalgae are widely used in wastewater treat-
ment. Chlorella sp. can grow and adapt to a polluted medium or
environment, show a fast growth rate, and assimilate nutrients
efficiently in wastewater. Chlorella sp. reduced COD and ammonia

https://doi.org/10.1016/j.matpr.2023.03.206
2214-7853/Ó 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0).
Selection and peer-review under responsibility of the scientific committee of the 3rd International Conference on Chemical Engineering and Applied Sciences.
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
by 74% and 72% [8]. In removing organic compounds and nutrients,
it is known that Chlorella sp. has a symbiotic relationship with
locally growing decomposing bacteria in wastewater [9]. Based
on this, nutrient removal in wastewater using bacterial and
microalgal co-cultures is an alternative and renewable approach
[10]. According to Han et al. [11], microalgae have the potential
to remove nutrients such as nitrogen content in wastewater with
high efficiency by utilizing these nutrients for their metabolic pro-
cesses to synthesize new cells, while bacteria can degrade organic
substances in wastewater [10].
The presence of natural bacteria in the wastewater medium also
increases the efficiency of organic substances and nutrient
removal, especially in tofu wastewater, through their interaction
with microalgae. Organic substances contained in tofu wastewater
can be broken down into macronutrients by decomposing bacteria
and used by microalgae [12]. From this process, bacteria produce
inorganic carbon, such as CO2, which microalgae can use for the
photosynthesis process. Bacteria can use the oxygen these microal-
gae produce from the photosynthesis process to accelerate the
decomposition process [13].
According to Dianursanti et al. [14], the tofu wastewater pro-
duced contains several organic materials, mainly protein and car-
bohydrates, that can be broken down into macronutrients with
the help of decomposing bacteria, which are then utilized by
microalgae for growth. The possibility of utilizing tofu wastewater
as a microalgae cultivation method is one way of solving the prob-
lem of tofu wastewater [15]. However, the tofu wastewater con-
centration given is known to affect the Chlorella sp. growth
because it is related to the ability of light to penetrate into the cul-
ture medium, and excess nutrient loading may inhibit microalgal
growth rates. Nevertheless, coping successfully with the high tur-
bidity and nutrient toxicity of wastewater often requires dilution
before using the wastewater as a growth medium [16].
One of the microalgae-based wastewater treatment technolo-
gies used to reduce pollutants in tofu wastewater is the Rotary
Algae Biofilm Reactor (RABR), with fixed film and suspended
growth systems identified as alternative bioreactors in treating
industry wastewater [17]. The Rotary Algae Biofilm Reactor is a
type of photobioreactor with a rotating biological contactor
(RBC)-based design to grow phototrophic microorganisms, mainly
microalgae, using a partially submerged rotating disk with growth
substrate attached to the surface of the disk media. Biofilm growth
is possible even in turbid wastewater systems because the RABR
rotates the biofilm in and out of the water, thereby exposing it to
both light and a nutrient source [18].
The RBC itself has several advantages due to its efficiency,
proper design, good gas exchange process, and high tolerance for
shock loading. Therefore, the RABR design is intended to maintain
these advantages by optimizing the growth of inorganic carbon-
based algae to form a mixed-culture biofilm consisting of microal-
gae and heterotrophic bacteria [19]. Based on how the RABR works,
microalgae can absorb nutrients in wastewater when the biofilm is
submerged. In contrast, when the biofilm is above the water’s sur-
face, microalgae can utilize CO2 in the atmosphere as well as sun-
light to support the photosynthesis process [20].
To test a biofilm-based approach to achieving these goals, a
rotating algal biofilm reactor (RABR) was designed. The RABR con-
sists of a cylinder provided with a growth surface partially sub-
merged in wastewater. The cylinder is rotated to alternately
expose the growth surface to the wastewater and the air. RABR is
one of the technologies that allow wastewater nutrient sources
to be utilized for microalgae-based systems that could not support
suspended microalgae growth due to turbidity, color, or water
depth limitations [21]. The purpose of this research was to analyze
the effect of tofu wastewater in different concentrations as a culti-
vation medium on the growth rate of biofilm and suspended Chlor-
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Materials Today: Proceedings 87 (2023) 263–271
ella sp. cells and also to determine the removal efficiency of COD
(Chemical Oxygen Demand) and ammonia (NH3) content in the
wastewater using a Rotary Algae Biofilm Reactor (RABR).
2. Materials and methods
This research was conducted at the Alga Research Center Labo-
ratory at the Faculty of Fisheries and Marine Sciences and the Lab-
oratory of Control and Prevention of Environmental Pollution at
the Faculty of Engineering, Riau University. Research on processing
tofu liquid waste aerobically using a batch system is carried out on
a laboratory scale. The stages of conducting this research include
seeding, acclimatization and operational with retention time vari-
ation. This study is to determine the efficiency of the removal of
wastewater parameters. The process used in this study is a batch
process by giving contact between tofu liquid waste samples and
mikroalga Chorella sp. in the reactor RABR for 5 days so that
changes in the quality of tofu liquid waste samples can be seen.
2.1. Sample collection
Tofu wastewater samples used as a growth medium were col-
lected from a local tofu industry in Jl. Cipta Karya, Tuah Karya Vil-
lage, Tampan District, Pekanbaru City, Riau. This area was selected
because the tofu industry did not treatment the liquid waste before
disposal of to the river. If left unchecked, this practice can reduce
the water quality. The sampling process of tofu wastewater is car-
ried out as much as 40 L according to Indonesian National Standard
(SNI) 6989.59-2008 by the grab sampling process.
For the main experiment, tofu wastewater was prepared
according to variations in the concentration of waste, namely
40%, 60%, 80%, and 100%, by diluting the tofu wastewater with dis-
tilled water, and as much as 13,500 ml was needed for each treat-
ment. The following steps are taken to prepare tofu wastewater:
 40% concentration = 5,400 ml of tofu wastewater + 8,100 ml of
fresh water
 60% concentration = 8,100 ml of tofu wastewater + 5,400 ml of
fresh water
 80% concentration = 10,800 ml of tofu wastewater + 2,700 ml of
fresh water
 100% concentration = 13,500 ml of tofu wastewater
2.2. Characteristics of tofu wastewater
One of the production processes is the tofu clumping process,
which results in a separate cloudy liquid, also known as ‘‘whey.”
According to Safrilia et al. [5], the presence of organic compounds
such as protein, carbohydrates, and fats can affect the levels of
organic substances and nutrients in tofu wastewater. Therefore,
an analysis of the initial characteristics of tofu wastewater, includ-
ing COD and NH3 parameters. The value of COD are critical in the
wastewater analyses, since the can show the amount of polluted
organic materials in the water system. Usually there is a good cor-
relation between COD and BOD value. Ammonia from tofu wastew-
ater in is ranging from 11 to 16 mg/l. The maximum allowable
wastewater ammonia is 5 mg/l. Ammonia is a group of nitrogen
compounds that are widely contained in wastewater, as toxic to
aquatic animals and harmful to aquatic ecosystems. COD and
NH3 was carried out to determine the initial concentration in each
treatment of tofu wastewater that would be used as a medium for
the cultivation of microalgae (Chlorella sp.). The results of the ini-
tial characteristic test of tofu wastewater based on Table 1 show
that tofu wastewater still has high levels of pollutants and exceeds
the quality standard, so further processing is needed before being
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
Table 1
Characteristics of tofu wastewater with variations in concentration.
Tofu wastewater Parameters (mg/L) Quality Standards
Concentration (mg/L)
COD NH3 COD NH3
40% wastewater 1028 11.87 300a 5b
60% wastewater 1510 13.70
80% wastewater 1975 14.95
100% wastewater 2466 15.56
a
Minister Regulation of Environment Republic of Indonesian Number 5 of 2014,
Attachment XLVII.
b
Minister Regulation of Environment Republic of Indonesian Number 5 of 2014,
Attachment XVIII.
discharged into water bodies. Microalgae can utilize pollutants in
tofu wastewater, consisting of organic compounds and nutrients,
as nutrients for their growth. It is known that using tofu wastew-
ater as an alternative medium for microalgal growth is one way
to solve the problem of tofu wastewater.
2.3. Seeding and acclimatization of Chlorella sp.
In this study, the microalga Chlorella sp. was obtained from the
Algae Research Center, Faculty of Fisheries and Marine Sciences,
Riau University, and was cultivated on Dahril Solution’s medium
to increase the number of cells until it reached the exponential
phase, with the number of cells reaching 1
106 cells [15]. Chlor-
ella sp. was cultivated with Dahril Solution medium, a nutrition
source that uses sunlight as a light source for photosynthesis and
cell division. The Dahril Solution used is an adequate medium with
nutrients that contain macronutrient elements such as nitrogen,
sulfur, potassium, phosphorus, and calcium, as well as micronutri-
ents such as iron, boron, manganese, zinc, cobalt, and copper,
which can increase the growth and metabolism of Chlorella sp. cells
[22]. Chlorella sp. was grown in 3.5 L of distilled water using
100 ml of Chlorella sp. culture and 400 ml of Dahril Solution med-
ium. The light source for this process comes from direct sunlight.
The cultivation process was conducted for 7 days, total number
of cells at the end of the process was 1.13
106 cells/mL, so it
has reached the exponential phase and will go through the
acclimatization process.
Acclimatization is a process that aims to adapt the microalgae
cells of Chlorella sp. to the tofu wastewater as a new growth med-
ium by mixing it with microalgae culture gradually so that the
microalgae can adapt to the characteristics of the wastewater. In
addition, acclimatization was also carried out for the formation
Fig. 1. Microalgae cells and indigenous bacteria microscopically after acclimatization process.
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Materials Today: Proceedings 87 (2023) 263–271
of biofilms on disk media. In this process, indigenous bacteria
found in tofu wastewater interact with microalgae to produce EPSS
(extracellular polymeric substances), which help in the process of
stable biofilm adhesion on the surface of the disk media [20].
Acclimatization is carried out gradually over 6–10 days until the
microalgal exponential phase reaches 1
106 cells/mL [15].
In this study, the acclimatization stage will go through two
stages with a working volume of 18 L. The initial stage is carried
out by mixing the culture results of the cultivation of the microal-
gae Chlorella sp. with tofu wastewater in a 50%:50% ratio. In the
first stage of the acclimatization process, Chlorella sp. reached the
exponential phase on day 6 with a suspension-based cell density
of 1.06
106 cells/mL and a biofilm-based cell density of
0.81
106 cells/mL. In the next stage, the results of the first
acclimatization stage can be mixed with tofu wastewater in a ratio
of 75% to 25%. On day 12, the optimum density of microalgae cells
in suspension and biofilm reached 1.11
106 cells/mL and
1.06
106 cells/mL, respectively.
Based on this, the microalgae Chlorella sp. can adapt and utilize
the nutrients contained in tofu wastewater for metabolism and cell
growth. In this acclimatization process, the indigenous bacteria
found in tofu wastewater can interact with microalgae to produce
EPSS (extracellular polymeric substances), which help in the pro-
cess of stable biofilm attachment on the surface of the disk media
[20]. Proteins, cations, and organic molecules contained in tofu
wastewater are concentrated on the surface of the immersed and
rotating disk media to support the growth of microorganisms such
as microalgae and bacteria in forming the biofilm layer [19]. In this
study, the identification of indigenous bacteria in tofu wastewater
showed that Bacillus sp. which are decomposing bacteria organic
substances in tofu wastewater and are proteolytic and can produce
protease enzymes to degrade proteins contained in tofu wastewa-
ter. This is also supported by the research conducted by Ken et al.
[23], which identified indigenous bacteria in tofu wastewater. It
was found that the genus Pseudomonas and genus Bacillus were
the most dominant bacteria. Asril et al. [24] also isolated indige-
nous bacteria with the genus Pseudomonas and Aeromonas in tofu
wastewater. The following observations on microalgae and indige-
nous bacteria in tofu wastewater microscopically after going
through the acclimatization stage can be seen in Fig. 1.
2.4. Experimental setup
In this research, the tool prepared is the RABR, which consists of
four reactors made of acrylic with the exact dimensions of
(length
width
height) 30
30
30 cm. This reactor is planned
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
Fig. 2. Design of Rotary Algae Biofilm Reactor.
to be able to process tofu wastewater with an effective volume of
18 L of wastewater. Each reactor for each research treatment is
given direct sunlight. The following research tool design can be
seen in Fig. 2.
The disk media is a place for microalgae biofilm to be attached,
so the disk media used must make it easier for microalgae to stick.
In this study, the disk media used was a thin round acrylic disk
with a diameter of 12 cm that was hydrophobic, so this media
made it easier for microalgae to adhere. The presence of hydropho-
bic properties is a combination of roughness characteristics, where
the rougher the surface of the medium, the higher the
hydrophobicity.
In this study, the main experiment was cultivating the micro-
alga Chlorella sp. on tofu wastewater medium in a RABR reactor
with a working volume of 18 L (18,000 ml). The RABR process con-
sists of a combination of suspended growth and attached growth
systems. A total of 13,500 ml of tofu wastewater was put into each
RABR reactor based on variations in treatment, namely variations
in the concentration of tofu wastewater of 40%, 60%, 80%, and
100%. In addition, microalgae suspension with a fixed concentra-
tion of 25% of the working volume (4,500 ml) was added [25]. All
treatment variations in this experiment were given lighting from
sunlight. During the processing, pH and temperature measure-
ments were carried out to observe the growing environmental con-
ditions. In this case, the initial cell count of the microalgae
suspension was calculated. The main trial time lasted for 0, 1, 3,
and 5 days.
2.5. Data analysis and processing
The parameters analyzed in this study were observations on the
efficiency of removal of COD and NH3 content and measurement of
the number of Chlorella sp. cells, pH, temperature, with variations
in the concentration of wastewater, and also the best contact time.
After observing for a span of 0, 1, 3, and 5 days, a parameter anal-
ysis was carried out to determine the level of the parameters after
going through processing that was adjusted to the applicable
Indonesian National Standard (SNI) in Table 2 below.
To determine the efficiency of decreasing the test parameters,
the following equation can be used:
Table 2
Pollutant parameter analysis.
Analysis Method/tools
Microalgae cell density Thomacytometer
pH SNI 06-6989.11-2004
Temperature Thermometer
COD SNI 6989.73-2009
NH3 SNI 06-6989.30-2005
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Materials Today: Proceedings 87 (2023) 263–271
Cin  Cef
Efficiency ð%Þ ¼
100 ð1Þ
Cin
where:
Cin = Influent concentration (mg/L).
Cef = effluent concentration (mg/L).
3. Results and discussion
3.1. Effect of tofu wastewater concentration on Chlorella sp. growth
During the processing, the number of microalgae (Chlorella sp.)
suspended in the reactor is counted every 24 h for five days. This
process was carried out to determine the growth pattern of Chlor-
ella sp. microalgae, which were cultivated on tofu wastewater
medium, were treated with variations in the concentration of
waste given. The growth of microalgae cells (Chlorella sp.) in cul-
ture, according to Hadiyanto & Azim [26], can be indicated by an
increase in the number of cells. In this study, to determine whether
there is an increase in the number of microalgae cells every day,
observations can be made with the help of a thomacytometer mea-
suring instrument and a light microscope.
Based on Fig. 3, it is known that each culture that grows with
various concentrations of tofu wastewater produces a different
value for the number of microalgae Chlorella sp cells. This indicates
that variations in the concentration of tofu wastewater influence
the growth of suspended Chlorella sp. cells. Based on the graph
below, it is known that from the beginning to the end of the con-
tact time, the 40% tofu wastewater concentration treatment
resulted in the highest Chlorella sp. cell number on day five at
2.18
106 cells/mL.
On day 0, the amount of suspension of microalgae Chlorella sp.
varied with variations in the concentration of tofu wastewater of
40%, 60%, 80%, and 100%, respectively, namely 8.40
105 cells/
mL, 8.70
105 cells/mL, 8.30
105 cells/mL, and 8.10
105/mL.
It could be observed that the microalgae cells on day 0 had not
yet experienced a growth process; a slight decrease in the number
of cells from the initial inoculum cell number was caused by the
dilution process when the suspension of the microalgae Chlorella
sp. added with tofu wastewater medium, and it is known that
the effect of tofu wastewater concentration has not been seen. This
follows the research of Mardatillah [27], where the number of
microalgae cells, Chlorella sp., on day 0 showed a slight decrease
due to the addition of wastewater and had not experienced a good
growth process. Until the first day, Chlorella sp. microalgae cells at
each concentration variation still experienced a lag phase where
Chlorella sp. was still adapting to the environment in the Rotary
Algae Biofilm Reactor (RABR), so the growth was not optimal. In
the early stages, the inoculated cells undergo chemical and physi-
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
Fig. 3. Graph of Chlorella sp. suspended cells number during treatment.
ological changes to readjust their metabolic activities so that they
can grow in new media.
After adjusting to the medium’s environmental conditions, the
Chlorella sp. cells will enter the exponential phase (log phase).
Chlorella sp. cells grew on tofu wastewater with a concentration
of 40%, and 60% of them experienced an exponential phase from
day 2 to day 5 so that they did not experience subsequent phases
during the processing. The lag phase was known to happen for a
short time because of the ability of microalgae to grow and adapt
well to utilize nutrients in tofu wastewater. This is influenced by
the acclimatization process carried out before the processing. Elys-
tia et al. [28] stated that microalgae cells that underwent acclima-
tization were physiologically more active and able to utilize more
nutrients in wastewater for growth and metabolism.
In the treatment of a 40% tofu wastewater concentration, the
highest cell growth occurred because the tofu wastewater con-
tained inorganic minerals in the form of ions from the decomposi-
tion of other microorganisms, such as bacteria in non-sterile tofu
wastewater, so that it was more easily absorbed and utilized by
Chlorella sp. cells optimally for growth. In addition, the amount
of nutrients present in tofu wastewater is proportional to the num-
ber of microalgae that utilize these nutrients to produce microal-
gae with the highest cell density [29]. Lu et al. [30] stated that
too high a nutrient concentration in wastewater could inhibit the
growth of microalgae. High concentrations of nutrients can cause
environmental conditions that do not support the growth of
microalgae. In this condition, the self-protection mechanism of
microalgae is activated, and its growth is inhibited. The high dilu-
tion level of 40% tofu wastewater concentration was found to be
better because it reduces the concentration of toxic substances
and also increases the clarity of the medium, giving a chance for
maximum light penetration as well as efficient photosynthetic
activities to occur so that the suspended-based productivity of
Chlorella sp. microalgae can occur optimally [16]. The chemical
equation for the utilization of carbon dioxide and nutrients in tofu
wastewater in the cell formation process can be shown as follows:
106 CO2 + 236 H2 O + 16 NH4þ +HPO4 2
!C106 H1841 O45 N16 P + 118O2 + 171H2 O + 14Hþ
The exponential phase was also experienced by the microalgae
cells of Chlorella sp. at concentrations of tofu wastewater of 80%
and 100%, but the number of cells was not as high as at concentra-
tions of tofu wastewater of 40% and 60%, and on the last day of pro-
cessing, it was observed that the cell division process did not occur
intensively. This indicates that the microalgae Chlorella sp. have
reached the end of the exponential phase and are beginning to
enter a decline in growth rate, which begins with slowing down
cell growth. According to Elystia et al. [28], the growth in the num-
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Materials Today: Proceedings 87 (2023) 263–271
ber of cells tends to be less due to the excess volume of tofu
wastewater, causing the presence of excessive nutrients so that it
becomes an inhibition factor for Chlorella sp. cells, causing the
growth of microalgae cells to be less than optimal.
In addition, based on Amalah et al. [29], tofu wastewater with a
higher concentration has higher turbidity so that it can inhibit light
entry into the medium. This, of course, can slow down the photo-
synthesis process and cause the microalgae’s growth rate to
decrease. The presence of impurities, such as remaining lumps of
tofu, and dissolved organic and inorganic materials, can inhibit
the absorption of light by microalgae. Ratnani et al. [31] also men-
tion that organic substances or suspended substances from tofu or
soybeans are scattered and dissolved, causing tofu wastewater to
resemble a cloudy emulsion.
The growth of microalgae cells Chlorella sp., based on the bio-
film in this study, was initiated by the formation of an initial bio-
film layer on the surface of the disc through an adsorption
process that occurred during the acclimatization process, resulting
in the microalgal Chlorella sp. biofilm-based growth that was char-
acterized by an increase in the number of cells and the thickness of
the biofilm on the disk media. This research shows that the con-
centration of tofu wastewater affects the number of Chlorella sp.
biofilm growth cells.
From Fig. 4, it can be seen that the number of microalgae cells of
Chlorella sp. attached on day 0 is the same as the number of
microalgae cells of Chlorella sp. in the second stage of the acclima-
tization process, where the variations in the concentration of tofu
wastewater are 40%, 60%, 80%, and 100%, respectively, namely
1.07
106 cells/mL, 1.06
106 cells/mL, 1.11
106 cells/mL and
9.8
105 cells/mL. On day 1, it was known that all the concentra-
tion variations had a slight increase in the number of cells. This
indicates that Chlorella sp. biofilm-based microalgae cells can adapt
well [26]. After going through the adaptation phase, the microalgae
cells of Chlorella sp. experienced an exponential phase on days 2–5
for tofu wastewater concentrations of 40% and 60% and on days 3–
5 for tofu wastewater concentrations of 80% and 100%. Based on
the observations made, the number of microalgae cells of Chlorella
sp. The highest biofilm-based concentration was found in the 40%
tofu wastewater concentration variation of 1.81
106 cells/mL,
and the lowest was in the 100% tofu wastewater concentration
with a cell count of 1.19
106 cells/mL.
At a 40% tofu wastewater concentration, the growth of microal-
gae cells (Chlorella sp.) based on biofilm is known to be directly
proportional to the growth of Chlorella sp. suspended cells.
Microalgae cells in the biofilm layer can absorb organic substances
and nutrients contained in tofu wastewater to perform their meta-
bolic processes. Applying the 80% submerged depth of the disk
causes a significant contact between the biofilm layer and the
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
Fig. 4. Graph of cell number of microalgae Chlorella sp. biofilm-based during
processing.
wastewater medium so that the cells of Chlorella sp. obtain suffi-
cient nutrients for the productivity of microalgal biomass [32]. In
this study, the tofu wastewater medium used had a non-sterile
condition where other microorganisms, such as decomposing bac-
teria, were present. The presence of bacteria in tofu wastewater
causes interactions between bacteria and microalgae that result
in forming a biofilm layer attached to the surface of the disk media
through a combination of EPS (extracellular polymeric substances)
produced by both. This type of biofilm is a combination of auto-
trophic and heterotrophic components suitable for wastewater
treatment applications because it quickly absorbs nutrients in pol-
luted water [33].
In this study, it is known that the concentration of tofu wastew-
ater treatment given can affect the number of microalgae cells
(Chlorella sp. attached) and the condition of the formed biofilm
layer. Based on the observations that have been made, the biofilm
layer at the 40% tofu wastewater concentration, the treatment disk,
was dominated by microalgae cells of green color that were
slightly cloudy and not too thick. This indicates that the high rate
of Chlorella sp. cells in this treatment was due to the unobstructed
diffusion of light into the biofilm layer so that the photosynthesis
process was not hampered [34]. Meanwhile, at the 100% concen-
tration of tofu wastewater, which is more concentrated, the
unsignificant growth rate of chlorella cells is assumed to be caused
by a more dominant bacterial biofilm layer marked by the presence
of white turbid spots and clumps on the surface of the biofilm
layer. This is supported by the statement of Boelee et al. [35],
where the white spots on the surface of the biofilm consist mostly
of bacteria, which are thought to be bound to each other by EPS
(extracellular polymeric substances) so that they can inhibit light
Fig. 5. Chlorella sp. biofilm on disk media (a) before treatment (b) after treatment at 40% concentration (c) at 100% concentration.
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Materials Today: Proceedings 87 (2023) 263–271
Fig. 6. Graph of COD removal efficiency during processing.
transmission into the microalgae biofilm layer and slow down
the photosynthesis process for the growth of Chlorella sp. cells
[36]. The microalgae biofilm attached to the disk media can be seen
in Fig. 5.
3.2. Tofu wastewater pollutants removal by Chlorella sp.
3.2.1. COD removal
Based on Fig. 6, it can be seen that during the 5 days of process-
ing, the best COD removal occurred at a 40% tofu wastewater con-
centration variation of 75.88%, with a decrease in the initial COD
concentration from 1,028 mg/l to 248 mg/l. Meanwhile, the lowest
COD removal occurred in 100% tofu wastewater concentration at
20.36%, removing the initial COD content of 2,466 mg/l to
1,964 mg/l. From the graph, it can be observed that the lower the
concentration of tofu wastewater given, the higher the removal
of COD levels. The graph below shows that the decrease in COD
levels is directly proportional to the length of contact time, where
the longer the contact time, the higher the removal of COD levels.
The treatment with a 40% concentration of wastewater is
known to be the optimal concentration of nutrients and can stim-
ulate the growth of microalgae so that higher COD levels can be set
aside. This is supported by the research of Saidu et al. [16], where
more dilution of wastewater causes the reduction of excess nutri-
ents and reduces the level of turbidity of the medium so that light
penetration occurs optimally and has an impact on the efficient
photosynthesis process. Fazal et al. [37] stated that the removal
of COD levels would increase along with the increase in processing
time. The increase in time during the growth period led to a higher
concentration of microalgae cells.
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
The decrease in COD levels in each treatment with variations in
the concentration of tofu wastewater indicates that microalgae can
utilize and assimilate organic compounds in tofu wastewater in the
metabolic processes of their cells. According to Widayat &
Hadiyanto [38], optimal microalgal growth is indicated by a greater
reduction in COD. Istirokhatun et al. [39] stated that the growth of
microalgae cells was inversely proportional to the COD content in
tofu wastewater. The greater the number of microalgae cells (in
suspension growth and attached growth), the lower the COD con-
tent would be.
Many species of microalgae, one of which is Chlorella sp., can
reduce COD levels through mixotrophic metabolism, where
organic carbon can be used in the presence of a light source [10].
Microalgae are mixotrophs that can assimilate CO2 and organic car-
bon simultaneously. Mixotrophic tofu wastewater treatment by
Chlorella sp. can effectively utilize nutrients and organic pollutants
in wastewater to efficiently support microalgal growth. In non-
sterile culture conditions, it is known that the presence of bacteria
in the tofu wastewater medium can increase the efficiency of
removing COD levels [36]. This is supported by the research con-
ducted to identify indigenous bacteria in tofu wastewater; the bac-
teria genera Pseudomonas and Bacillus were found to be the most
dominant bacteria. It is known that the two genera of bacteria act
as bacteria that decompose organic substances and can produce
protease enzymes to degrade proteins in tofu wastewater [23].
In this study, tofu wastewater treatment using RABR became
more effective due to the mutualistic interaction that occurs
between bacteria and microalgae. Based on research by Hongyang
et al. [40], tofu wastewater contains highly organic substances,
such as monosaccharides, oligosaccharides, vitamins, organic
acids, amino acids, lipids, whey protein, isoflavones, saponins, P,
Ca, Fe, and other nutrients. Mutualism symbiosis can occur when
bacteria decompose complex organic compounds to produce sim-
pler compounds such as organic acid molecules, amino acids, car-
bon dioxide, and ammonium, which can be utilized by microalgae
for their growth [36]. Under sufficient light conditions, microalgae
cells can produce oxygen for the environment when the metabolic
process takes place. Various metabolic products are excreted by
microalgae, such as carbohydrates, polysaccharides, proteins, and
others; some of these products can be collected by bacteria and
assimilated and mineralized into ions that can be absorbed by
microalgae. The following interactions between microalgae and
bacteria in wastewater can be seen in Fig. 7.
Tofu wastewater treatment using suspended-based microalgae
will not be as effective as tofu wastewater treatment using a com-
Fig. 7. Mutualism in the interaction of microalgae and bacteria.
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Materials Today: Proceedings 87 (2023) 263–271
bination of suspended-based and biofilm microalgae cell growth.
This is supported by research by Wang et al. [41], which states that
the presence of microalgae biofilms is better at removing organic
matter than only using microalgae suspensions. Mutjaba et al.
[10], in their research, also stated that the best removal of COD
and nutrients was shown in bacterial microalgae co-cultures with
a combination of suspension-based and attached growth.
3.2.2. Ammonia removal
Based on Fig. 8, the best NH3 removal efficiency was found in
variations in the concentration of tofu wastewater by 40% on the
5th day, namely 80.45%, with a decrease in the initial NH3 level
from 11.87 mg/l to 2.32 mg/l. On the other hand, the lowest NH3
removal occurred at 100% concentration treatment with 25.19%
efficiency, setting aside the initial NH3 concentration of
15.56 mg/l to 11.64 mg/l. The graph above shows that the concen-
tration of tofu wastewater and contact time affect the removal of
NH3 levels. The allowance for NH3 levels in tofu wastewater is
known to be higher with the length of contact time. This is sup-
ported by the research of He et al. [8], where ammonia levels
decreased until the end of the processing time.
The concentration of tofu wastewater can affect the intensity of
light that can enter the culture medium, so it also affects the pho-
tosynthesis process as well as the total amount of nutrients that
can be assimilated by microalgae [29] he amount of nutrients in
the 40% concentration variation of tofu wastewater is not exces-
sive, making the culture medium suitable and optimal for the
growth of microalgae Chlorella sp.
[37]. In addition, the interaction of microalgae with indigenous
bacteria is known to increase the efficiency of NH3 removal in tofu
wastewater. This is supported by research conducted by Liang et al.
[42], showing that co-cultures of Chlorella sp. and B. licheniformis
showed higher ammonia removal efficiency compared to removal
by microalgae alone. This is because the bacteria B. licheniformis
can support the growth of Chlorella sp. On the other hand, in the
treatment of tofu wastewater concentration, 100% of the medium
culture conditions contained a nutrient composition that was
unsuitable for the growth of Chlorella sp., so the growth of microal-
gae is not optimal. This causes higher ammonia levels since
microalgae do not completely assimilate it. It is known that an
excess concentration of ammonium can be toxic to microalgae
growth, especially if it is in alkaline conditions [43].
The number of microalgae cells in Chlorella sp. is known to be
related to the removal of NH3 levels contained in tofu wastewater.
It can be seen that there has not been a significant allowance from
S. Elystia, Fakhriyah Hanifa Mazaya Nasution and A. Sasmita
Fig. 8. Graph of NH3 removal efficiency during processing.
day 0 to day 1 of the growth of microalgae Chlorella sp.; this is due
to the growth of microalgae Chlorella sp., which is still adjusting
their cell conditions with tofu wastewater media, so that less
NH3 can be absorbed by microalgae [26]. From day 3 to day 5, there
was an increase in NH3 removal, which was higher, as seen by the
decrease in NH3 concentration. The decrease in NH3 content in tofu
wastewater indicates that the microalgae Chlorella sp. can utilize
ammonia as a source of nutrients in their growth.
According to Mutjaba et al. [10], nitrogen removal in wastewa-
ter is more efficient with a combination of attached and suspended
microorganism growth. The biofilm layer is known to have a posi-
tive influence on the activity of microalgae during the process of
nutrient absorption. According to Zhang et al. [36], protein, which
is one of the organic nitrogen sources, is the primary nitrogen
source in the tofu wastewater medium. Organic nitrogen contained
in tofu wastewater is mostly decomposed with the help of decom-
posing bacteria into simpler compounds such as amino acids.
Amino acids, which result from protein reshuffle, will be oxidized
to ammonia, nitrogen (NH3), and carboxyl compounds.
On microalgae culture medium, in general microalgae will
absorb ammonia (NH3) in tofu wastewater first until it runs out
through glutamine synthesis, then it will absorb nitrate (NO3),
because the process of transport and assimilation of ammonium
ions by microalgae cells requires less energy compared to transport
and assimilation of nitrate. (NO3). Nitrate and nitrite will be
reduced first by nitrate reductase and nitrite reductase. Nitrate
reductase will catalyze 2 electrons from NADH to be converted into
nitrite. Nitrite will then be converted into NH+4 by nitrite reductase
and ferredoxin (Fd), where in this reaction 6 electrons will be
transferred. All forms of inorganic nitrogen will be converted into
the form of NH+4 to be further converted into amino acids in
microalgae cells, where this amino acid will react with L-
glutamine to produce ATP which functions in the photosynthesis
process [44].
4. Conclusion
Based on the research that has been done, it can be concluded
that the optimum tofu wastewater concentration for microalgae
medium growth can be seen in the treatment of tofu wastewater
at 40% concentration, resulting in the highest suspended and
attached cell growth numbers of 2.18
106 cells/mL and
1.81
106 cells/mL, as well as the highest COD and NH3 removal
efficiency values during the processing of tofu wastewater using
RABR technology, namely 75.88% and 80.45% with the best contact
time obtained on the fifth day. The final concentrations of COD and
NH3 tofu wastewater after processing are 248 mg/l and 2.32 mg/l,
respectively, and are known to have met the quality standards for
wastewater for soybean (tofu) processing businesses or activities
in the Minister Regulation of the Environment of the Republic of
Indonesia Number 5 of 2014 in Appendix XVIII. The cultivation of
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Materials Today: Proceedings 87 (2023) 263–271
Chlorella sp. in tofu waste medium has high potential because it
offers a wide range of environmental and economic benefits as a
sustainable strategy to treat wastewater and produce high-value
products from microalgae.
CRediT authorship contribution statement
Shinta Elystia: Conceptualization, Methodology, Writing – orig-
inal draft, Investigation, Data curation. Fakhriyah Hanifa Mazaya
Nasution: Formal analysis, Writing – review & editing, Visualiza-
tion. Aryo Sasmita: Visualization, Supervision.
Data availability
Data will be made available on request.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The authors would like to express their gratitude to the Alga
Research Centre Laboratory at the Faculty of Fisheries and Marine
Sciences and the Laboratory of Control and Prevention of Environ-
mental Pollution at the Faculty of Engineering—both at the Univer-
sity of Riau—for their support in this research.
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