This study aimed to produce fungal biomass from agro-industrial by-products for later use as a bioflocculant in the Spirulina harvesting. The production of fungal biomass from Aspergillus niger was carried out in submerged fermentation, using media composed of wheat bran and/or potato peel. Fungal biomass was used as a bioflocculant in Spirulina cultures carried out in closed 5 L reactors and 180 L open raceway pond operated in batch and semi-continuous processes, respectively. Fungal biomass was able to harvest Spirulina platensis cultures with

1. Bioresource Technology 322 (2021) 124525
Available online 10 December 2020
0960-8524/© 2020 Elsevier Ltd. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
Harvesting of Spirulina platensis using an eco-friendly fungal bioflocculant
produced from agro-industrial by-products
Mateus Torres Nazari a
, César Vinicius Toniciolli Rigueto b
, Alan Rempel a
, Luciane
Maria Colla a,b,*
a
Postgraduate Program in Civil and Environmental Engineering (PPGEng), Faculty of Engineering and Architecture (FEAR), University of Passo Fundo (UPF), Passo
Fundo, RS, Brazil
b
Postgraduate Program in Food Science and Technology (PPGCTA), Faculty of Agronomy and Veterinary Medicine (FAMV), University of Passo Fundo (UPF), Passo
Fundo RS, Brazil
H I G H L I G H T S G R A P H I C A L A B S T R A C T
• An eco-friendly fungal-assisted bio­
flocculation process was developed.
• Fungal biomass is an efficient bio­
flocculant of Spirulina platensis.
• Spirulina biomass was totally harvested
in some experimental conditions.
• Aspergillus niger was cultivated using
agro-industrial by-products.
A R T I C L E I N F O
Keywords:
Bioflocculation
Aspergillus niger
Submerged fermentation
Spirulina platensis
Cyanobacteria
Sustainability
A B S T R A C T
This study aimed to produce fungal biomass from agro-industrial by-products for later use as a bioflocculant in
the Spirulina harvesting. The production of fungal biomass from Aspergillus niger was carried out in submerged
fermentation, using media composed of wheat bran and/or potato peel. Fungal biomass was used as a bio­
flocculant in Spirulina cultures carried out in closed 5 L reactors and 180 L open raceway pond operated in batch
and semi-continuous processes, respectively. Fungal biomass was able to harvest Spirulina platensis cultures with
efficiencies between 90% and 100% after 2 h of sedimentation in some experimental conditions. Efficiencies
higher than 80% were achieved in most tests without pH adjustment during bioflocculations, which shows that
the developed method is a promising alternative to traditional Spirulina harvesting techniques. Above all, the
development of an eco-friendly fungal-assisted bioflocculation process increases the sustainability of Spirulina
biomass for different applications, especially biofuels.
* Corresponding author at: Postgraduate Program in Civil and Environmental Engineering (PPGEng), Faculty of Engineering and Architecture (FEAR), University of
Passo Fundo (UPF), Passo Fundo, RS, Brazil.
E-mail address: lmcolla@upf.br (L.M. Colla).
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
https://doi.org/10.1016/j.biortech.2020.124525
Received 24 October 2020; Received in revised form 3 December 2020; Accepted 5 December 2020

2. Bioresource Technology 322 (2021) 124525
2
1. Introduction
Several high value-added bioproducts are obtained from microalgal
biomass, such as polysaccharides, proteins, long-chain polyunsaturated
fatty acids, phenolic compounds, vitamins, pigments, among others
(Andrade et al., 2018). Besides, microalgae can be used to treat
emerging contaminants (Rempel et al., 2020), nutrients (Rezvani et al.,
2018), dyes, and heavy metals (Chu and Phang, 2019) present in
aqueous media. However, the cultivation of microalgae is limited by
economic aspects, being the harvesting one of the challenges for
obtaining algal biomass for biotechnological applications, since the size
of the cells (micrometers) coupled with high colloidal stability in the
culture medium make the harvesting process more expensive and
difficult (Khoo et al., 2020; Yin et al., 2020).
The methods commonly used for microalgae harvesting are centri­
fugation, coagulation-flocculation, filtration, flotation, or a combination
of these techniques (Sakarika and Kornaros, 2019). However, in physical
processes, such as centrifugation, a large use of energy is required, while
in chemical methods (e.g. coagulation-flocculation), even with a high
recovery rate, there may be contamination of the biomass, which can
affect the steps following the harvesting and until limiting the recycling
of the culture medium (Salim et al., 2011; Yin et al., 2020).
In this context, the harvesting of microalgae by bioflocculation is a
promising alternative due to its characteristics of economy, simplicity of
operation, and efficiency (Nazari et al., 2020; Salim et al., 2011; Zhou
et al., 2013). This technique is based on the same principle as conven­
tional flocculation, but biological organisms are used as flocculating
agents, such as bacteria, algae, fungi, among others (Alam et al., 2016;
Nazari et al., 2020; Ummalyma et al., 2017).
The use of fungi as bioflocculants is an efficient strategy for micro­
algae harvesting processes, which occurs mainly through two main
mechanisms: the interaction and subsequent aggregation of microalgal
cells due to the difference in surface charges between organisms (Alam
et al., 2016; Chen et al., 2018). Studies report excellent efficiencies of
microalgae harvesting by fungal-assisted bioflocculation, using Asper­
gillus fumigatus (Bhattacharya et al., 2019), Penicillium sp. (Chen et al.,
2018), Aspergillus niger (Alrubaie and Al-Shammari, 2018), Mucor circi­
nelloides (Gu et al., 2017) and Aspergillus oryzae (Zhou et al., 2013).
However, the mentioned studies used Chlorella species, which pre­
sents differences in shape and size compared with the Spirulina, being
the bioflocculation of Spirulina still little explored. The publications until
now involve experimental studies performing inductions in Spirulina
cultivation to mediate autoflocculation (Markou et al., 2012; Vergnes
et al., 2019), and the use of chitosan and eggshell (Lai et al., 2019;
Shurair et al., 2019) and guar gum (Jana et al., 2018) as bioflocculants of
Spirulina. Oliveira et al. (2019) was the only work that used fungus
(Aspergillus niger pellets) as a bioflocculant, in which Spirulina maxima
was one of four genus evaluated in initial bioflocculation experiments.
This highlights the gap in the bioflocculation of Spirulina platensis,
mainly assisted by filamentous fungi.
Another advantage of the use of fungi as bioflocculant agents is
related to their cultivation since it is possible to use wastes or agro-
industrial by-products as substrates (Kreling et al., 2020). This
approach is considered a green technology, as these wastes that have
little or no commercial value can cause negative environmental impacts
if managed improperly (Sadh et al., 2018). Besides, the use of these
residues and agro-industrial by-products in the composition of culture
media allows valorization, contributing to the development of circular
economy processes with greater sustainability. Thus, the aim of this
study was to produce fungal biomass from agro-industrial by-products
and use it as a bioflocculant of Spirulina platensis cultivated in open and
closed bioreactors.
2. Material and methods
2.1. Production of Aspergillus niger by submerged fermentation (SmF)
Submerged fermentation was carried out using the filamentous
fungus Aspergillus niger DAOM (Colla et al., 2015). The strain was kept in
test tubes with potato dextrose agar (PDA) at 4 ◦
C. The preparation of
fungal inocula for the cultures was carried out by adding 5 mL of a 0.1%
solution of sterile Tween in test tubes containing the fungus Aspergillus
niger, obtaining a suspension of fungal spores. From this suspension, 0.5
mL was transferred to a Petri dish containing sterile PDA. The dishes
were incubated at 30 ◦
C for 6 days, being the spores used to inoculate the
submerged fermentation media.
Wheat bran and potato peel were used as components of the culture
media to obtain Aspergillus niger biomass, using the following experi­
mental conditions: a) Wheat bran (WB) as the main component of the
culture medium with the addition of 0.5% NaNO3 as a nitrogen source
and initial fermentation pH of 6.0 b) Potato peel and wheat bran (PP +
WB) as main components of the culture medium, 1% NaNO3 and pH 5.0
(these experimental conditions showed the best results of fungal
biomass production in 2 days of fermentation in preliminary tests, data
not showed). In both fermentations, the amount of residue used to
compose the culture medium was calculated in order to provide 15 g L-1
of starch. In the fermentation with PP + WB, 50% of the starch was
obtained from PP and 50% from WB. It was considered that 18% of the
WB is consisted of starch (dry basis) (Xie et al., 2008) and that 74% of
the carbohydrates in the potato peel (wet basis) are starch (Javed et al.,
2019).
A suspension of 10% (w/v) of WB and PP + WB in distilled water was
cooked at 100 ◦
C for 30 min, followed by filtration to remove the
insoluble solids. Then, the nitrogen source was added and the pH was
adjusted, with subsequent sterilization of the medium at 121 ◦
C for 20
min. 100 mL volumes of sterile media were added in 250 mL sterile
flasks. Medium was inoculated with an aseptic transfer of 2 mycelial
disks of fungus (1 cm diameter) from the preparation of the inoculum in
Petri dishes. The media were incubated for 2 days in an orbital shaker
(TE-421 model, Tecnal, Brazil) at 30 ◦
C, at 0,97g (120 rpm). After 2 days,
the fermented media was centrifuged (Centrifuge 5810, Eppendorf,
Germany) at 2,588g (3,500 rpm) for 15 min. The solid fraction was
tested as a bioflocculant in the harvesting of Spirulina platensis cultures.
Part of the fungal biomass was dried for further chemical
characterization.
2.2. Cultivation of Spirulina platensis
Spirulina platensis was cultivated in Zarrouk medium (Zarrouk, 1966)
with nutrients depletion, at a Zarrouk concentration of 20% (w/v) and
Spirulina initial concentration of 0.2 g.L− 1
. Cultivations were carried out
in two modes in order to verify if they would affect the harvesting ef­
ficiencies and biochemical composition of biomass: a) in semi-
continuous mode in an open raceway pond (300 L), with a useful vol­
ume of 180 L, and agitation provided by rotating blades (0.35 m.s− 1
); b)
in batch mode in 5 L transparent rigid plastic bottles, with aeration from
air injection by compressors (U-2800, Boyu, China). The cultures were
carried out in a greenhouse of transparent film, temperature range be­
tween 20 ◦
C and 30 ◦
C, and luminosity provided by natural light. Cul­
tures were maintained until the stationary growth phase, when cell
density was stable, reported as the one that results in the greatest
accumulation of reserve carbohydrates by Spirulina when linked to the
depletion of nutrients in the medium (Magro et al., 2018).
Culture samplings were performed three times a week to determine
pH by potentiometry and the microalgal biomass concentration through
a pre-established relationship between absorbance at 670 nm and dry
weight (presented in Supplementary material), both in triplicate.
M.T. Nazari et al.

3. Bioresource Technology 322 (2021) 124525
3
2.3. Bioflocculation
The bioflocculation tests were carried out in two steps. In Step 1, wet
fungal biomass was used to perform the bioflocculation of Spirulina
cultures. This step was evaluated by a complete factorial design (22
with
3 central points), being the studied variables the pH (4, 6, and 8) and the
concentration (w/v) of wet bioflocculant (2.5%, 5%, and 7.5%) added in
100 mL of microalgal media. The ratio fungi:algae (dry basis) was
determined considering the fungal biomass moisture of 90%.
The pH of the medium was adjusted and then the wet fungal biomass
was added. A mechanical stirrer (713 model, Fisaton, Brazil) was used to
perform a rapid mixing at 200 rpm (2g) for 1.5 min, followed by slow
mixing at 20 rpm (0.016g) for 10 min (Sossella et al., 2020). After the
slow mixing, the medium was kept at rest in three sedimentation times
(30 min, 2 h, and 20 h), and samples were taken after each time to
determine the Spirulina biomass concentration of the liquid not sepa­
rated in the bioflocculation. Harvesting efficiency was determinate by
Eq. (1). Bioflocculation tests without pH adjustment were performed as
controls.
Harvesting efficiency (HE, %) =
(C0 − Cf )
C0
× 100 (1)
where,
HE: Harvesting efficiency (%);
C0: Initial concentration of Spirulina (g.L-1
);
Cf: Final concentration of Spirulina (g.L-1
).
In Step 2, the influence of the variation of pH during the submerged
fermentation of fungal biomass production over the harvesting effi­
ciency, was investigated. The initial fermentation pHs were 4, 5, 6, and
7, and the PP + WB were used as a substrate. In these tests, the micro­
algae bioflocculation was accomplished using 0.5%, 1.5%, and 2.5% of
wet fungal, without adjusting the pH in the bioflocculation process,
using Spirulina cultivated in the open raceway pond (180 L).
2.4. Analytical determinations
2.4.1. Characterization of wheat bran and potato peel
Wheat bran and potato peel were characterized for moisture, protein,
lipid, and ashes (fixed mineral residue), according to the methods
described by AOAC (2000). The percentage of total carbohydrates was
obtained by difference from the other components.
2.4.2. Characterization of Spirulina and fungal biomasses
Biomasses of Aspergillus niger and Spirulina platensis were dried at
50 ◦
C until constant weight in an air circulation oven (TE-394-1 model,
Tecnal, Brazil) to determine the carbohydrate (Dubois et al., 1956) and
protein content (AOAC, 2000). These biomasses were also qualitatively
characterized by infrared analysis with Fourier Transform (FTIR). The
samples were subjected to spectroscopic determination in the infrared
region (500–4,000 cm− 1
) (Cary 630 FTIR, Agilent, United States) using
the total attenuated reflectance (ATR) technique.
2.5. Data treatment and statistical analysis
Data resulting from the factorial designs were evaluated for signifi­
cance and estimated effects of the main and interaction variables (p <
0.05). The differences between the averages of the tests were evaluated
by analysis of variance (ANOVA), followed by the Tukey test, with a
95% confidence level.
3. Results and discussion
3.1. Characterization of substrates and biomasses
Table 1 shows the characterization of wheat bran and potato peel
used as a substrate in submerged fermentation for fungal biomass
production.
Water content and carbohydrates were the main components of po­
tato peel (79.48% and 18.01%, respectively). Carbohydrates were the
major fraction of wheat bran, with around 68.85%, due to the lower
water content compared to potato peel. After carbohydrates, the major
component was proteins. Carbohydrates and proteins are essential for
microbial growth since they are a source of carbon and nitrogen,
respectively. Wheat bran is an excellent substrate to be used for Asper­
gillus niger growth (Kreling et al., 2020). The characterization of wheat
bran (Table 1) is similar to the studies by Kreling et al. (2020), while the
potato peel is similar to the values reported by Arapoglou et al. (2010).
Fungal and microalgal biomasses were analyzed by FTIR, considered
an adequate method for the qualitative characterization of the chemical
composition of microbial cells (Shapaval et al., 2014). In the Supple­
mentary Material 2, the Figures show the FTIR spectra of the Aspergillus
niger and Spirulina platensis biomasses.
When comparing fungal biomasses cultivated on different substrates
(Fig. A1 in the Supplementary material 2), the peak with higher in­
tensity is found at 1019 cm− 1
and 1338 cm− 1
for the Aspergillus niger
grown in WB and PP + WB. The range of 1000–1350 cm− 1
is charac­
terized by the presence of amine groups (C-N stretching), being the two
main peaks of the biomasses located in this region (Mungasavalli et al.,
2007).
Peaks between 1000 and 1300 cm− 1
and 900–1185 cm− 1
may
indicate the presence of C–O stretching of COOH and polysaccharides,
respectively, where the Aspergillus niger cultivated on wheat bran
showed the peak with the highest intensity (Gáplovská et al., 2018;
Mungasavalli et al., 2007; Volesky, 2007), which is according to the
carbohydrate content (almost 3 times higher) of the Aspergillus niger
biomass cultivated on a medium composed only of WB concerning the
fungus cultivated on PP + WB (Table 2).
Aspergillus niger cultivated in PP + WB showed a different peak in the
range of 1300–1375 cm− 1
, reported by the presence of sulfamide bonds
(S–
–O), as well as proteins, lipids, and phosphate compounds
(1185–1485 cm− 1
) (Mungasavalli et al., 2007; Tralamazza et al., 2013).
The higher peak intensity observed in the Aspergillus niger biomass
Table 1
Chemical composition of agro-industrial by-products used as a substrate in SmF.
Wheat bran Potato peel
Wet basis Dry basis Wet basis Dry basis
Moisture (%) 11.70 ±
0.47
– 79.48 ±
0.80
–
Lipids (%) 3.25 ± 0.05 3.68 ± 0.06 0.29 ± 0.11 1.41 ± 0.53
Proteins (%) 11.96 ±
0.22
13.54 ±
0.25
1.43 ± 0.18 6.35 ± 0.80
Ashes (%) 4.24 ± 0.03 4.81 ± 0.03 0.80 ± 0.09 3.90 ± 0.44
Carbohydrates
(%)
68.85 ±
0.70
77.97 ±
0.79
18.01 ±
1.00
87.77 ±
4.87
Mean ± sd (n = 3).
Table 2
Characterization of microalgal and fungal biomasses.
Cultivation specifications Proteins
(%)
Carbohydrates
(%)
Spirulina
platensis
Batch (closed reactor 5 L) 27.42 ±
0.22c
46.19 ± 2.54b
Semi-continuous (open
raceway pond 180 L)
33.51 ±
0.08d
49.12 ± 2.26b
Aspergillus
niger
Wheat bran 21.87 ±
0.19a
51.05 ± 1.95b
Potato peel + Wheat bran 26.05 ±
0.19b
19.39 ± 4.67a
Mean ± sd (n = 3). Equal letters in the columns indicate that there was no
statistical difference (p < 0.05) by the Tukey test.
M.T. Nazari et al.

4. Bioresource Technology 322 (2021) 124525
4
cultivated on wheat bran (Fig. A1, Supplementary material 2) may be
related to a greater amount of negative charges, which tends to decrease
its interaction with Spirulina cells and, thus, reduce the harvesting
efficiency.
Nithya et al. (2019) report that hydroxyl, amide, carboxylate,
phosphoryl, nitrate, and phenolic groups are prevalent in Spirulina
species. The main difference observed between these biomasses of
Spirulina obtained from the two cultivation modes (Fig. A2, Suple­
mentary material 2) occurred between 1350 and 1450 cm− 1
, with
emphasis on the peak located at 1407 cm− 1
for Spirulina cultivated in
batch mode. According to Venkatesan et al. (2012), the range between
1435 and 1405 cm− 1
presents CH2 bending vibration, associated with
the presence of carbonyl compounds (negatively charged), which have a
high potential to avoid the agglomeration of Spirulina (El-Naggar et al.,
2020; Kanchana et al., 2011). This correlates with the best biomass
harvesting results obtained in open cultivation, that will be presented
latter (Table 3).
Table 2 shows the protein and carbohydrate contents of the bio­
masses of Spirulina platensis obtained in the two cultivation modes per­
formed (Step 1), as well as Aspergillus niger cultivated on a medium
composed of wheat bran and potato peel + wheat bran, in the first step
of this study.
Both Spirulina platensis cultures resulted in a high accumulation of
intracellular carbohydrates (Table 2). Commonly, Spirulina has high
protein content (46–63%), but an increase in its carbohydrate content
can be achieved through nutritional and environmental stresses during
cultivation, such as nutrient limitation, light availability, harvest phase,
aeration, among others, which increases its viability for bioethanol
production (Becker, 2007; Magro et al., 2018). The change in the levels
of carbohydrates and proteins of microalgae is beneficial, as these
compounds can be used in different applications (Rempel et al., 2020).
Results in Table 2 demonstrate that Spirulina cultivated in open raceway
pond (180 L) had higher protein and carbohydrate contents, being more
advantageous compared to closed reactor (5 L) cultivation, since high
levels of proteins and carbohydrates are important for the production of
feed, biofuels and biofertilizers (Becker, 2007; Rempel et al., 2020).
Regarding the composition of fungal biomasses, the cultivation in
wheat bran produced biomass with higher carbohydrate content, similar
to that reported by Volesky (1987), differing from the Aspergillus niger
cultivated on potato peel + wheat bran (PP + WB) (p < 0.05). This
difference in the composition of fungal biomasses may have been
responsible for the different efficiencies obtained in the bioflocculation
tests presented below since the biomass charges can vary according to
the carbohydrates and proteins content, as also demonstrated in the
FTIR spectra.
Fungal biomass used as bioflocculant in Step 2 were also character­
ized qualitatively by FTIR. Figure B, in the Suplementary material 2
shows the FTIR spectra of the Aspergillus niger biomass cultivated on PP
+ WB at initial pHs (4, 5, 6, and 7) of SmF. The fungal biomass (Step 2)
presented a similar spectrum for all biomasses from SmF with different
initial pH, since all the characteristic peaks are presented, with only a
few transmittance variations, considered normal in the ATR-FTIR
spectra. This similarity between the fungal biomasses spectra may
explain the same harvesting efficiencies obtained in the bioflocculation
experiments of Step 2 performed with Aspergillus niger biomasses culti­
vated at pH 4, 5, 6, and 7 (Fig. 2).
Table 3
Harvesting efficiencies (HE) of Spirulina platensis cultures using fungal biomass as bioflocculant (Step 1).
Culture Experiment pH Bioflocculant
(%)
Fungi:algae
ratio (w/w)
Wheat bran (WB) Potato peel + Wheat bran (PP + WB)
HE (%) 0.5
h
HE (%) 2 h HE (%) 20 h HE (%) 0.5
h
HE (%) 2 h HE (%) 20
h
Open raceway pond
(180 L)
1 4 2.5 2:1 94.07 ±
0.23eA
98.74 ±
0.09gB
100 ±
0.02eC
99.55 ±
0.03fA
100 ±
0.07eB
100 ±
0.03cB
2 8 2.5 2:1 79.18 ±
0.04cA
87.85 ±
0.07eB
94.30 ±
0.19dC
97.74 ±
0.13eA
99.11 ±
0.10cB
100 ±
0.04cC
3 4 7.5 6:1 89.38 ±
0.24dA
95.55 ±
0.04fB
98.23 ±
0.02eC
100 ±
0.01gA
100 ±
0.04eA
100 ±
0.01cA
4 8 7.5 6:1 68.01 ±
0.11aA
76.49 ±
0.11aB
87.97 ±
0.13ªC
96.40 ±
0.04 dB
98.48 ±
0.10bC
87.35 ±
0.05aA
5 6 5 4:1 79.25 ±
3.29cA
84.50 ±
0.05cB
90.26 ±
2.43abC
95.98 ±
0.24cA
99.84 ±
0.02 dB
100 ±
0.03cB
6 6 5 4:1 78.59 ±
0.13cA
84.05 ±
0.08bB
90.69 ±
0.18bC
96.69 ±
0.05dA
99.90 ±
0.04deB
100 ±
0.03cC
7 6 5 4:1 79.52 ±
0.41cA
86.90 ±
0.18 dB
91.81 ±
0.23bcC
93.70 ±
0.24bA
100 ±
0.02eB
100 ±
0.05cB
Control* 10.11 5 4:1 74.87 ±
0.14bA
84.74 ±
0.07cB
93.30 ±
0.06cdC
84.04 ±
0.12aA
93.72 ±
0.03aB
93.85 ±
0.04bB
Closed reactor (5 L) 1 4 2.5 2.4:1 77.74 ±
0.25gA
87.01 ±
0.15fB
95.51 ±
0.08gC
88.69 ±
0.12fA
96.76 ±
0.03gB
99.39 ±
0.03eC
2 8 2.5 2.4:1 43.45 ±
0.33bA
65.51 ±
0.16bB
78.34 ±
0.20bC
46.52 ±
1.34aA
73.49 ±
0.05bB
84.41 ±
0.04bC
3 4 7.5 7.3:1 65.27 ±
0.06fA
79.57 ±
0.15eB
87.03 ±
0.06fC
91.63 ±
0.31gA
93.65 ±
0.13fB
99.16 ±
0.05eC
4 8 7.5 7.3:1 21.94 ±
0.19aA
34.99 ±
0.59aB
64.10 ±
0.28aC
58.52 ±
0.30bA
63.99 ±
0.06ªB
66.67 ±
0.66aC
5 6 5 4.9:1 63.28 ±
0.28eA
75.42 ±
0.13 dB
84.69 ±
0.07dC
91.25 ±
0.03gA
93.18 ±
0.11eB
96.76 ±
0.03dC
6 6 5 4.9:1 61.90 ±
0.18dA
74.21 ±
0.22cB
86.51 ±
0.15eC
78.54 ±
0.22cA
92.42 ±
0.06 dB
96.87 ±
0.10dC
7 6 5 4.9:1 62.25 ±
0.24dA
75.14 ±
0.46 dB
84.36 ±
0.11cdC
87.05 ±
0.07eA
92.91 ±
0.31eB
96.65 ±
005dC
Control* 11.08 5 4.9:1 50.75 ±
0.25cA
73.68 ±
0.16cB
84.15 ±
0.14cC
80.85 ±
0.29dA
86.72 ±
0.24cB
89.96 ±
0.10cC
Mean ± sd (n = 3). Equal letters in the columns and in the lines indicate that there was no statistical difference (p < 0.05) by the Tukey test.
*Control: Just fungal biomass (5%) without pH adjustment.
M.T. Nazari et al.

5. Bioresource Technology 322 (2021) 124525
5
3.2. Spirulina platensis harvesting by Aspergillus niger biomass-assisted
bioflocculation
The concentration of Spirulina platensis biomass was 1.25 g.L-1
and
1.03 g.L-1
and the pH of 10.11 and 11.08 in 22 days of cultivation for
open raceway pond and closed reactor, respectively, when the bio­
flocculation process was realized. Table 3 shows the harvesting effi­
ciencies of Spirulina platensis biomass obtained in the bioflocculation
experiments of the Step 1 in three sedimentation times (0.5 h, 2 h, and
20 h).
In general, it can be seen in Table 3 that the harvesting efficiency
increases with a longer sedimentation time in all tests. Fungal biomass
cultivated on a medium composed of potato peel and wheat bran was
more efficient for Spirulina platensis harvesting, compared to the biomass
obtained in the cultivation using only wheat bran as substrate. This
higher efficiency was observed for both Spirulina cultivation modes.
However, Spirulina biomass from semi-continuous (180 L) cultivation
was harvested with higher efficiency than batch (5 L) culture, which can
be related to lower pH value and better fungi:algae ratio of open race­
way pond cultivation. In two hours of sedimentation, some tests with PP
+ WB showed 100% of harvesting efficiency for Spirulina cultivated in
open raceway pond. Besides, the increase of fungal biomass resulted in a
decrease of harvesting performance, which means that 2.5% (w/v) was
the optimum concentration of fungal biomass (in wet basis) used as
bioflocculant tested in Step 1. Based on Table 3, it is possible to define
that: a) Spirulina platensis cultivation in open raceway pond; b) Asper­
gillus niger cultivated on PP + WB; and c) two hours of sedimentation; are
the best conditions considered for bioflocculation of Spirulina platensis
assisted by Aspergillus niger biomass in view of the obtained efficiencies
and production aspects, such as time and volume of process.
Experiments 1 and 3 showed the highest separation efficiency
(Table 3). These two tests had in common the pH adjustment at the
lowest levels tested (pH = 4). pH reduction results in the insertion of
more positive charges in the media and, as microalgae have a negative
charge (Rashid et al., 2013), this may have favored the interaction be­
tween microorganisms and, consequently, in the bioflocculation pro­
cess. According to Chen et al. (2018), pH more acidic is a major factor to
achieve high separation efficiencies, since it induces cells to have greater
contact and generate aggregates in the presence of H+
ions. Another
important aspect is that the pH adjustment to values lower than the pH
of the Spirulina platensis cultivation results in an increase in millivoltage
(Sossella et al., 2020), which improves the biomass harvesting. Gu et al.
(2017) reported that charge neutralization is the main mechanism in the
bioflocculation between algae and fungi.
Efficiencies above 80% were achieved in most experiments with the
addition of 5% Aspergillus niger biomass without pH adjustment (control
tests). Thus, it is possible to infer that the fungal biomass has a charge
opposite to the Spirulina since bioflocculation based on the algae-fungi
interaction requires that the fungus be metabolically active and that it
has an opposite charge to perform the algal biomass separation (Praja­
pati et al., 2016).
Surface charge of the Aspergillus versicolor biomass, determined from
the measurement of the zeta potential, ranged from +15.1 (pH 2) to
− 35.5 mV (pH 8), and the zero charge point was observed at pH 3,5 (Das
et al., 2007). The same behavior was reported by Luo et al. (2019), who
found that between the pH range of 2 to 3.3, the zeta potential of the
fungus Pleurotus ostreatus was positive and that below 3.3 there was
greater interaction with Chlorella sp., which increased the harvesting
efficiency.
Negative charges in the Spirulina platensis-maxima culture medium
decrease with pH reduction, since at pH 8, 6, and 4, the cultivation of
this microalgae presented a zeta potential around − 37 mV, − 33 mV, and
− 28 mV, respectively, achieving charge neutralization at pH 2 (Kőnig-
Péter et al., 2014). This is related to the efficiencies shown in Table 3,
where the best biomass recovery percentages were obtained in the ex­
periments with the lowest pH value (p = 4). However, high efficiencies
achieved in the other tested pH values and, even in the tests without pH
adjustment, show that the fungal biomass acted as a bioflocculant agent
of Spirulina platensis.
Table 4 presents the levels of significance (p) and estimated effects
(p < 0.05) of the studied factors in the bioflocculation tests with two
different fungal biomasses on the harvesting efficiency of Spirulina pla­
tensis in two cultivation modes (Step 1).
The factors studied in the bioflocculation performed with fungal
biomass from fermentation with wheat bran showed significant main
effects (p < 0.05) in the two modes of Spirulina platensis cultivation
(Table 4), with an interaction effect in closed reactor cultivation.
Aspergillus niger cultivated on PP + WB medium, when applied as a
bioflocculant in Spirulina cultivation in open raceway pond, showed pH
as a significant variable, whereas both main and interaction effects were
significant for harvesting efficiency in the closed reactor. Thus, for the
Spirulina cultivation carried out in open raceway pond it is possible to
define that the pH adjustment of the media to 4 increases the harvesting
efficiency for both fungal biomasses. Regarding the bioflocculant con­
centration, it is possible to define that the lowest level tested (2.5%) is
the best condition to harvest Spirulina cultures in open raceway pond,
since this variable was significant for the Aspergillus niger cultivated on
wheat bran and, although not significant (p > 0.05) for the fungus
cultivated on PP + WB, a smaller amount of fungal biomass for the same
efficiency obtained represents lower costs.
In all cases where the pH and the bioflocculant concentration were
significant (Table 4), the effects of these variables were negative on the
harvesting efficiency, which means that when the lower levels of pH and
bioflocculant concentration passed to the higher levels, there was a
significant reduction in the bioflocculation process efficiency (p < 0.05).
There was a significant interaction between the main variables in the
experiments carried out in closed reactor, which are shown in Fig. 1.
Fig. 1 shows that both tested bioflocculant concentrations result in
lower harvesting efficiencies when the pH is at its higher level. There­
fore, pH 4 and 2.5% of bioflocculant are the best conditions to perform
the proposed bioflocculation method to harvest Spirulina platensis by
Aspergillus niger biomass.
The presence of carboxylic groups, amines and phosphate results in
negative surface charges on the cell wall of most microalgae, while the
fungal surface groups are protonated at a highly acidic pH, which results
in a positive charge on their hyphae (Oliveira et al., 2019). This charge
difference is fundamental for the interaction between microalgae and
fungus to achieve high harvesting efficiency. Pleurotus ostreatus pellets
were tested by Luo et al. (2019) to harvest Chlorella sp. These authors
observed that at the lowest tested pH value (pH = 3) there was the
greatest harvesting efficiency (about 65% in 150 min), which
Table 4
Significance levels (p) and estimated effects of the variables studied in the first
step of bioflocculation on the harvesting efficiency in two hours of
sedimentation.
Cultivation Factor WB1
PP + WB2
p Effect p Effect
Open
raceway
pond
(180 L)
(1) pH 0.010330 − 14.9755 0.004464 − 1.20535
(2)
Bioflocculant
(%)
0.041709 − 7.2740 0.061482 − 0.31065
(1) × (2) 0.116891 − 4.0834 0.061482 − 0.31065
WB3
PP + WB4
Closed
reactor
(5 L)
(1) pH 0.000364 − 33.0324 0.000211 − 26.4585
(2)
Bioflocculant
(%)
0.001102 − 18.9786 0.003698 − 6.3077
(1) × (2) 0.002972 − 11.5402 0.014181 − 3.1955
WB: Wheat bran; PP + WB: potato peel + wheat bran; WB1
: R2
: 0.9859; R2
adjusted: 0.9577; PP + WB2
: R2
: 0.9938; R2
adjusted: 0.9815; WB3
: R2
: 0.9995;
R2
adjusted: 0.9986; PP + WB4
: R2
: 0.9997; R2
adjusted: 0.9991.
M.T. Nazari et al.

6. Bioresource Technology 322 (2021) 124525
6
corroborates with our results. Although we used other microorganisms
in the bioflocculation process, it was possible to show that Aspergillus
niger biomass was able to act as a bioflocculant of different Spirulina
platensis cultures, demonstrating that the interaction between these two
microorganisms is able to efficiently harvest Spirulina cells.
Fungal biomass cultivated on PP + WB showed higher harvesting
efficiency than that cultivated on WB (Table 3). This could be related to
the initial pH of the SmF (initial pH 5 for PP + WB; initial pH 6 for WB).
In order to investigate in more detail the influence of the submerged
fermentation pH on the harvesting efficiency of Spirulina platensis, bio­
flocculation experiments were carried out with fungal biomass produced
in SmF with an initial pH of 4, 5, 6, and 7. Fig. 2 presents the harvesting
efficiencies of Spirulina platensis using Aspergillus niger biomass culti­
vated on PP + WB, without pH adjusting of the microalgae media, after
two hours of sedimentation.
Fig. 2 shows that greater harvesting efficiencies (~80–90%) were
obtained when the initial pH of fungal cultures was 4, 5, and 6 (p <
0.05). Bioflocculant concentration used influenced the harvesting effi­
ciencies only at the initial pH 7 of cultivation, with the highest har­
vesting efficiency obtained in the lowest concentration of bioflocculant
tested (0.5%) (p < 0.05). Thus, the pH of the SmF was not a determining
factor in the effectiveness of the bioflocculation process, if the concen­
tration of the bioflocculant is maintained in 0.5%. This can reinforce
that the substrate of the cultivation of Aspergillus niger may be the main
responsible for the highest harvesting efficiencies obtained by fungal
biomass cultivated on PP + WB (Table 3).
Chen et al. (2018) reported that the bioflocculation performed with
pellets of Penicillium sp. was able to harvest ~98% of Chlorella sp.
biomass, in 2.5 h, with a 1:2 ratio of fungi:algae. In Step 1 of the present
work, high harvesting efficiencies were obtained in 2 h of sedimentation
in the Spirulina platensis cultivation carried out in open raceway pond
(180 L) using a 2:1 fungi:algae (dry basis) ratio (2.5% of fungus), 4:1
(5% of fungus) and 6:1 (7.5% of fungus), considering an average fungus
moisture of 90% (Table 3). In Step 2 (Fig. 2), it was decided to decrease
the fungi:algae ratio, where higher harvesting efficiencies were achieved
in the fungi:algae ratio of 0.6:1 (0.5% of fungus), 1.8:1 (1.5% of fungus)
and 3:1 (2.5% of fungus), respectively.
In view of the fact that microalgal biomass can be used for subse­
quent saccharification and fermentation and, as enzymes and yeasts can
be inhibited for the presence of metals in the biomass, the bio­
flocculation overlaps conventional coagulation-flocculation from the
point of view of sustainability. In this sense, bioflocculation is consid­
ered an efficient and low-cost technology for harvesting microalgae
(Ummalyma et al., 2017). In the case of this work, it was possible to
produce an eco-friendly fungal bioflocculant from agro-industrial by-
products, which adds value to these residues and promotes aspects of
circular economy in the microalgae production for different purposes.
4. Conclusions
Aspergillus niger biomass cultivated on agro-industrial by-products in
2 days of SmF was able to act as a bioflocculant agent of Spirulina,
obtaining harvesting efficiencies above 90% in two hours of sedimen­
tation. It is important to note that in experiments where only the
insertion of fungal biomass (without pH adjustment) was performed
there was harvesting efficiency above 80%, which shows the viability of
the bioflocculation method proposed. Therefore, an efficient and eco-
friendly fungal-assisted bioflocculation strategy was developed to har­
vest Spirulina platensis, which could increase the sustainability of
microalgae cultivations for different applications.
CRediT authorship contribution statement
Mateus Torres Nazari: Conceptualization, Investigation, Data
curation, Writing - review & editing. César Vinicius Toniciolli
Rigueto: Data curation, Writing - review & editing. Alan Rempel:
Fig. 1. Interaction plots between the bioflocculation assay variables of the
batch cultivation of Spirulina using fungal biomass cultivated on WB (3a) and
PP + WB (3b).
Fig. 2. Harvesting efficiency (mean, n = 2) of Spirulina biomass according to
the initial pH of SmF (p < 0.05).
M.T. Nazari et al.

7. Bioresource Technology 322 (2021) 124525
7
Investigation, Writing - review & editing. Luciane Maria Colla:
Conceptualization, Writing - review & editing, Supervision, Resources,
Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This study was financed in part by the Coordenação de Aperfeiçoa­
mento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2020.124525.
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