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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

Cultivation of Chlorella vulgaris in non-sterilized tofu

wastewater anaerobic digestion effluent

U Hamidah1*, N Ajijah1, Widyarani1 and N Sintawardani1

1
Research Unit for Clean Technology (LPTB), National Research and Innovation
Agency Republic of Indonesia (BRIN)
Komplek LIPI Bandung, Bandung City, West Java 40135 Indonesia

*E-mail: umih001@brin.go.id

Abstract. Microalgae cultivation on a large scale requires large amounts of water and nutrients;
this causes the cultivation process to be less economical. Tofu wastewater anaerobic digestion
effluent (TWADE) from an anaerobic wastewater treatment plant (WWTP) contains nutrients
that can be used as an alternative medium for microalgae cultivation to reduce cultivation costs.
This study aimed to utilize non-sterilized TWADE as a low-cost cultivation medium for
Chlorella vulgaris. Microalgae cultivation was carried out for ten days using TWADE 50% and
100%, with and without NaHCO3 addition. Walne medium was used as a positive control, and
tap water was used as a negative control. All mediums were diluted and prepared without
sterilization, only cloth-filtered. The physicochemical characterization of non-sterilized
TWADE showed that TWADE contained nitrogen, phosphate, potassium and some trace
minerals such as Fe, Cu, B, Mo, which were potential nutrients for microalgae growth. The
results revealed that Chlorella vulgaris cultivated in TWADE had higher cell counts, protein,
chlorophyll a, chlorophyll b and carotenoids than the negative control. However, microscopic
observations found some protozoa, which was suspected of causing Chlorella vulgaris growth
disorders. In general, TWADE has potential as an alternative cultivation medium, but further
studies are required to find an easy and inexpensive strategy for contaminant removal.

Keywords: alternative medium; Chlorella vulgaris; tofu wastewater; tofu wastewater anaerobic
digestion effluent (TWADE); without sterilization

1. Introduction
The Ministry for Research and Technology of Indonesia reported that in 2010, there were around 84,000
micro-and-small-scale (MSS) tofu factories with a total production capacity of 2.56 million tons/year of
soybean in Indonesia [1]. A sample of untreated tofu wastewater from Bandung City, Indonesia,
contained 15.9 g/L of chemical oxygen demand (COD) with a low pH of 3.5 [2]. A high-performance
anaerobic digestion system can degrade up to 95% of dissolved COD in wastewater, resulting in an
effluent that can be discharged to the environment [3]. For example, a three-staged anaerobic system
was stable enough to overcome the low pH of tofu wastewater [2]. A wastewater treatment plant
(WWTP) has been developed and implemented in Giriharja Hamlet, Sumedang, Indonesia to treat tofu
whey from nine MSS tofu factories [4]. The WWTP consists of a multistage fixed-bed reactor with an
anaerobic digestion system and could reduce tofu whey's COD by more than 90% without disrupting
the nitrogen and phosphate contents [5].

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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

Wastewater from tofu production contains high organic matters that might pollute the environment
if not appropriately treated. Protein is the major organic constituent of tofu wastewater [6]. Anaerobic
digestion removes organic matters (C) but not nutrients (N and P); other processes are usually applied
to meet the stringent effluent criteria [7][5]. As a result, degradable organic matter is converted into
methane (CH4) and carbon dioxide (CO2), while water and most nutrients remain. Thus, the effluent
from anaerobic digestion contains a large amount of water, organic matter, nitrogen, phosphorus, and
other micronutrients [8]. Due to its abundance and richness in nutrients, wastewater can be a cost-
effective medium for microalgae cultivation.
Many types of microalgae can grow in wastewater. However, Chlorella sp. and Scenedesmus sp. are
the most common microalgae used for cultivation due to their rapid cell growth compared with other
microalgae cells [9]. Moreover, it can remove ammonia, total nitrogen, total phosphate and produce a
significant amount of biomass under a condition with high carbon dioxide levels [10]. Therefore,
Chlorella vulgaris and Scenedesmus sp. can manage waste, produce environmentally friendly and
economical renewable energy, and manage CO2 emission better than other plants [11]. Microalgae can
utilize inorganic carbon, predominantly CO2, in photoautotrophic mode as their primary carbon source.
In aqueous solutions, gaseous CO2 dissociates into bicarbonate (HCO3−) and carbonate (CO32−) ions
depending on the pH, with the precise equilibrium subjects to temperature, cation concentration and
salinity [12].
Chlorella sp. is a unicellular algal with a circular shape [13], containing high protein, chlorophyll,
carotene, vitamin, antioxidant, antimicrobial, and other nutrients. It can also grow in the poor medium
and degrade ammonia, nitrite, nitrate, and phosphate [14]. Cultivating Chlorella sp. using wastewater
can provide several advantages, including the low cost of the operation and energy requirements,
removing heavy metals and xenobiotic substances, the possibility of recycling assimilated nitrogen and
phosphorus into algae biomass as fertilizer, and the safety because of the natural ecosystems [15].
Many researchers have investigated microalgae-based treatment processes to remove nutrients from
a wide variety of wastewater. However, few were concerned with culturing microalgae in original
wastewater without sterilization and nutrient removal [16]. Another major challenge that still limits the
application of microalgae to treat wastewater is the non-sterile environment associated with the process.
Therefore, researchers should be encouraged to co-culture natural microalgae communities and other
microorganisms such as yeasts, fungi and bacteria. Co-culturing aims to create stable communities that
perform predictably whilst filling all ecological niches to limit the potential for contamination and
culture crashes [17].
Research by Luo [18] demonstrated that Scenedesmus sp. could grow rapidly in 10% effluent of
cattle manure anaerobic digestion diluted with sewage secondary sedimentation tank effluent, both
without sterilization. However, nutrient additives for the fast growth of microalgae were necessary. Ding
et al. [16] investigated the feasibility of cultivating microalgae in dairy farm wastewater without
sterilization. The wastewater was diluted 20, 10 and 5 times before being applied to cultivate Chlorella
sp., and the results showed good ammonia, phosphorus and COD removals. Simsek and Sisman-Aydin
[19] used non-sterilized textile wastewater as a potential source for culturing green microalgae
Golenkinia radiata. This species may potentially be used in both bioremediations of textile wastewater
and as a biomass wastewater-fed to obtain biofuel in the future.
This research aimed to investigate the feasibility of cultivating microalgae Chlorella vulgaris in non-
sterilized tofu wastewater anaerobic digestion effluent (TWADE) from the anaerobic WWTP in
Giriharja, Sumedang, Indonesia. In addition, the influence of medium variation with and without
NaHCO3 (as inorganic carbon source) on pH, cell number, protein, chlorophylls a and b and carotenoids
contents were investigated.

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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

2. Materials and Methods

2.1. Tofu wastewater anaerobic digestion effluent

Samples of tofu wastewater anaerobic digestion effluent (TWADE) were obtained from an anaerobic
wastewater treatment plant (WWTP) treating tofu wastewater located in Giriharja Hamlet, Sumedang,
West Java, Indonesia. The physicochemical characterization of TWADE was performed at the
laboratory of BALITSA (Balai Penelitian Tanaman Sayuran) Lembang, West Java, Indonesia; certified
by the Indonesian accreditation committee (KAN, Komite Akreditasi Nasional) and ISO 9001. The
parameters measured and methods/equipment used were: pH (pH meter), total solids (gravimetric),
organic total solids (gravimetric), ash contents (gravimetric), C-organic (Kurmies, spectrophotometer),
total nitrogen (Kjeldahl), phosphate (spectrophotometer), potassium (flame photometer), micronutrients
and heavy metals (AAS).

2.2. Cultivation medium

TWADE 50% and 100% were prepared with and without NaHCO3 addition as an inorganic carbon
source. For treatment with NaHCO3, the pH was adjusted to 8.5. Walne medium was used as a positive
control, and tap water was used as a negative control. All mediums were cloth-filtered without
sterilization. The composition of Walne medium followed Ajijah et al. [5]. The compositions of the
various medium used in this experiment are presented in Table 1.

Table 1. Variation of cultivation medium.

Tap water Walne TWADE NaHCO3 Microalgae
Parameter
(mL) (mL) (mL) (g) (mL)
(-) control 1140 - - - 60
(+) control - 1140 - - 60
TWADE 50% 570 - 570 - 60
TWADE 100% - - 1140 - 60
TWADE 50% + NaHCO3 570 - 570 1.05 60
TWADE 100% + NaHCO3 - - 1140 1.05 60

2.3. Microalgae cultivation

Chlorella vulgaris stock culture (7200 mg/L biomass in Walne broth medium) was obtained from Balai
Besar Perikanan Budidaya Air Payau (BBPBAP) Jepara, Indonesia. Each medium (Table 1) was
inoculated with Chlorella vulgaris stock culture at 5% v/v. PET cylinders (2000 mL total volume, 1200
mL working volume) were used as photobioreactors. The cultures were cultivated at room temperature,
under artificial illumination using Philip Tube Light 18W (photoperiod 12:12 h L/D cycle), and aerated
using an aquarium pump (Resun LP-100) for ten days. The growth curve was made based on the cell
number data to determine the growth phase of Chlorella vulgaris. The cell number's measurement was
carried out using the haemocytometer method with four replications. Protein was measured according
to the Lowry method [20]. In addition, chlorophyll a, chlorophyll b, and total carotenoids content were
quantified according to Dere et al. [21]. The analysis was performed in triplicate; the data presented are
the average results.

3. Results and Discussions

The physicochemical characteristics of tofu wastewater anaerobic digestion effluent (TWADE) used in
this experiment are presented in Table 2. TWADE had a pH of 8.4-9.0, which was suitable for Chlorella
vulgaris growth. He et al. [22] found that Chlorella vulgaris could grow seven times in a week at a pH
of 7–10.4. Non-sterilized TWADE contained 450 mg/L of total nitrogen, 190 mg/L of phosphate, and

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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

740 mg/L of potassium, which were potential nutrients for microalgae growth. Moreover, TWADE had
some trace minerals such as Fe, Cu, B, Mo, which were the required micronutrients for microalgae
growth.

Table 2. Physicochemical characteristics of tofu wastewater-anaerobic digestion effluent

Parameter Value Parameter Value
Physical parameters Macronutrients
pH 8.4 – 9.0 C-organic 130 mg/L
TS (Total Solids) 4001 mg/L N-total 450 mg/L
OTS (Organic total solids) 1546 mg/L Phosphate 190 mg/L
Ash 2455 mg/L Kalium 740 mg/L
Micronutrients Heavy metals
Fe total 4.8 mg/L Hg <0.02 mg/L
Cu 7 mg/L Pb <0.01 mg/L
Zn 0 mg/L Cd <0.01 mg/L
B 0.3 mg/L As 4.35 mg/L
Mo <0.03 mg/L Cr <0.01 mg/L
Ni 0.05 mg/L

During observation, the pH in all treatments increased except in tap water as a negative control
(Figure 1). The addition of NaHCO3 to TWADE slightly increased the pH compared to treatment
without NaHCO3. Li et al. [23] reported that low concentration NaHCO3 was an excellent carbon source
for Chlorella vulgaris. On the other hand, high concentration NaHCO3 was an excellent carbon source
capable of controlling protozoa and stimulating lipid accumulation of another green alga, i.e., Neochloris
oleoabundans. However, in our results, 1 g/L NaHCO3 with pH adjustment could increase the pH
(Figure 1) but insufficient to increase Chlorella vulgaris growth (Figure 2).

10

pH

7
0 1 2 3 4 5 6 7 8 9 10
Observation time (day)

Tap water Walne medium TWADE 50%

TWADE 100% TWADE 50% + NaHCO3 TWADE 100% + NaHCO3

Figure 1. pH condition during observation time in various mediums.

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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

3.E+07

2.E+07

2.E+07
Cell numbers
(cells/mL)
1.E+07

5.E+06

0.E+00
0 2 4 6 8 10
Observation time (Day)

Tap water Walne medium

TWADE 50% TWADE 100%
TWADE 50% + NaHCO3 TWADE 100% + NaHCO3

Figure 2. The growth curve of Chlorella vulgaris on various mediums based on cell count data using a
haemocytometer.

Ajijah et al. [5] reported that Chlorella vulgaris could not grow in undiluted sterile TWADE
containing 613.5 mg/L COD. On the other hand, this study shows that Chlorella vulgaris could grow
with non-sterilized TWADE as a medium. Moreover, the growth of Chlorella vulgaris in TWADE 100%
exceeded positive control. TWADE 100% experienced the most significant increase on day-6 with
2×107 cells/mL, suddenly decreased to 3×106 cells/mL (day-8), then slightly increased to 8×106 cells/mL
on day-10. On day-10, TWADE 100%+NaHCO3 showed the least number of cells (3×106 cells/mL), but
still higher than the negative control (2x106 cells/mL).

25

20

15
Chlorophylls a-b and
carotenoids contents
(µg/gfw)
10

0
Chl a-b Car Chl a-b Car Chl a-b Car Chl a-b Car Chl a-b Car Chl a-b Car
Tap water Walne medium TWADE 50% TWADE 100% TWADE 50% + TWADE 100%
NaHCO3 + NaHCO3

Observation time (Day-10)

Figure 3. Chlorophylls a-b and carotenoids contents in various mediums.

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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

Chlorophylls and carotenoids are natural pigments known for their various health benefits for humans
and various applications in diverse industries. Microalgae are a rich source of carotenoids and other
metabolites among various microbial alternatives and received special attention [24]. Chlorophylls and
carotenoids extraction can be done before lipid extraction to produce efficient and profitable processes
[25]. Figure 3 shows that after ten days of cultivation, Chlorella vulgaris grown in various TWADE
mediums produced higher chlorophylls a and b content than the positive and negative controls.
Carotenoids contents in TWADE 50%, TWADE 50%+NaHCO3 and TWADE 100%+NaHCO3
mediums were 19.3 µg/gfw, 20.8 µg/gfw, and 18.1 µg/gfw respectively, slightly higher than in the
Walne medium (13 µg/gfw).
The differences in chlorophylls contents among previous studies were probably caused by
differences in intensity, type, and regime of light provided. The differences could also occur because of
different nitrogen compositions in the medium, which affected the synthesis process of chlorophyll [26].
The chlorophylls and carotenoids were thought to align with the number of microalgae. Higher and
equivalent levels of chlorophyll and carotenoids to positive controls found in TWADE medium show
the potential of TWADE to produce chlorophyll as a by-product of the organic and nutrient removal
process.

500

400

300
Protein content
(mg/L)
200

100

0
Tap water Walne medium TWADE 50% TWADE 100% TWADE 50% + TWADE 100%
NaHCO3 + NaHCO3

Observation time (Day-10)

Figure 4. Protein content of Chlorella vulgaris in various mediums.

Previously, Ajijah et al. [5] reported that Chlorella vulgaris cultivated in the sterilized Walne
medium on day-10 had a protein content of 272.83 mg/L. In this study, however, Chlorella vulgaris
cultivated in the non-sterilized Walne medium only contained 57.8 mg/L of protein, lower than the
previous study. Figure 4 shows that TWADE 100% showed the highest protein content of Chlorella
vulgaris (409.8 mg/L), meaning higher biomass of Chlorella vulgaris also resulted in higher protein
content. Various cultivation conditions and microalgal strains influence the pigments, lipid, protein, and
carbohydrate composition [27].
Our qualitative microscopic data found several types of protozoa that was suspected to prey on the
microalgae and inhibit the increase of microalgae cell number. Unfortunately, quantitative data could
not be obtained due to equipment limitations. Nevertheless, our results showed that cloth-filtration as
medium preparation without sterilization could not prevent contamination such as protozoa, which
disrupted the microalgae's growth. Predatory zooplankton that consumes microalgae is a major

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GREENVC 2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1017 (2022) 012030 doi:10.1088/1755-1315/1017/1/012030

ecological problem in large-scale cultivation—the presence of predators causes a "pond crash", which
leads to a devastating loss of microalgal biomass in open ponds. Currently, in-depth knowledge about
predators of microalgal cultures is very limited. Therefore, a collaborative research effort for quality
control and quality assurance microalgal pond diagnostics is urgently required for the timely
implementation of grazer mitigation strategies [28].

4. Conclusion
The results of this study suggest that tofu wastewater anaerobic digestion effluent (TWADE) is a
potential alternative for microalgae cultivation medium, as shown by an increase in the number of
Chlorella vulgaris cells. After ten days of cultivation, Chlorella vulgaris grown in various TWADE
mediums could produce carotenoids, chlorophyll a and chlorophyll b higher than the negative control.
TWADE with NaHCO3 (1 g/L) addition and pH adjustment (8.5) was able to increase the pH of the
medium but could not increase the growth of Chlorella vulgaris. However, TWADE without
sterilization (cloth-filtered only) is susceptible to the contamination that interferes with microalgae
growth.

Acknowledgement
Deep appreciation and thank you for Neng Nur Mujahidah ( Universitas Jenderal Achmad Yani),
Yustika Desti Yolanda (Universitas Pendidikan Indonesia) and Kelompok Pengrajin Tahu Giriharja
Sumedang for many helps.

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