Bioresource Technology 294 (2019) 122167

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bioflocculation of cyanobacteria with pellets of Aspergillus niger: Effects of T

carbon supplementation, pellet diameter, and other factors in biomass
densification
Helena R. Oliveira, Isabelli D. Bassin, Magali C. Cammarota
⁎

Department of Biochemical Engineering, School of Chemistry, Federal University of Rio de Janeiro, Avenida Athos da Silveira Ramos, 149, Bloco E, Sala 203, Cidade
Universitária, 21941-909, Rio de Janeiro, RJ, Brazil

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: One of the hurdles of renewable energy production from photosynthetic microorganisms is separating the
Bioflocculation biomass from water in cultures. Bioflocculation with filamentous fungus Aspergillus niger, an alternative low-cost
Biomass densification method used for such separation, was studied with four cyanobacteria. Cocultures with Spirulina maxima and
Cyanobacteria Synechococcus subsalsus resulted in bioflocculation efficiencies up to 94%, while with Anabaena variabilis and
Synechococcus subsalsus
Anabaena siamensis bioflocculation did not occur. S. subsalsus was selected to evaluate the effect of cyano-
Aspergillus niger
bacterial initial concentration, fungal:cyanobacterial ratio, carbon supplementation, and pH on biomass densi-
fication. Bioflocculation efficiencies up to 98% in 48 h were obtained with fungal:cyanobacterial ratio 1:5 and
carbon supplementation. Despite the lower efficiency (54%), the highest concentration factor of S. subsalsus
suspension (62.8 – from 0.9 to 56.5 g/L) was obtained with ratio 1:5 without supplementation. This result was
attributed to the smaller pellet diameter (2.5 mm) and indicated that lower pellet growth is better for biomass
densification.

1. Introduction promising applications owing to their high biomass productivity and

simple nutritional requirements. In addition, agricultural lands and
Photosynthetic microorganisms such as cyanobacteria and micro- clean water are not necessary, which helps increase the interest in these
algae are important due to carbon fixation, mitigating the effects of biomasses. Furthermore, cyanobacteria can be used for renewable en-
carbon dioxide build up in the atmosphere. These organisms have ergy production, such as biodiesel, biohydrogen, and biogas (Cheng

⁎
Corresponding author.
E-mail address: christe@eq.ufrj.br (M.C. Cammarota).

https://doi.org/10.1016/j.biortech.2019.122167
Received 21 July 2019; Received in revised form 17 September 2019; Accepted 18 September 2019
Available online 20 September 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
H.R. Oliveira, et al.
Table 1
Conditions of preliminary bioflocculation tests conducted with the four cyanobacteria species and conditions adopted in tests to evaluate effects on bioflocculation of
S. subsalsus.
Coculture conditions Preliminary Bioflocculation Tests
Cyanobacterium Anabaena siamensis, Anabaena variabilis,
Spirulina maxima, Synechococcus subsalsus
a
Fungal:cyanobacterial ratio 1:1
Organic matter concn. RFMb
pH without adjustmentd
Cyanobacterium initial concn.e 0.345–0.614
Time of coculture 24 h
a
On dry weight basis.
b
Residual fungal medium.
c
Supplementation percentages of the original fungal culture medium concentration.
d
pH was the result of mixing the fungal pellets with the cyanobacteria suspension.
e
g TSS/L.
et al., 2019).
However, one of the main hurdles of industrial processes based in
microalgal and cyanobacterial biomass is the difficult downstream
processing of the culture, especially due to the high quantity of water,
as cultures have low biomass concentration (about 0.5 g/L, achieving
5 g/L in photobioreactors) (Vandamme et al., 2013). Harvesting of
cyanobacteria and microalgae can add up to 20–30% of total produc-
tion costs (Chen et al., 2018). Therefore, the optimization of harvesting
technologies is essential for large-scale production of biofuels and
biogas (Prajapati et al., 2016). Biomass harvesting is difficult because of
colloidal stability of cell suspension, due to highly negative surface
charge and small cell size (2–20 μm) (Wan et al., 2015).
Many separation processes have been used to remove water from
algae biomass. These processes may be physical (sedimentation, flota-
tion, filtration, centrifugation, and physical flocculation), chemical
(chemical flocculation), or biological (microbial bioflocculants-asso-
ciated bioflocculation, microorganism-associated bioflocculation, and
self-flocculation). While biological methods are close to ideal in terms
of efficiency, costs, environmental impacts, and biomass contamina-
tion, they are usually strain specific and are still in the bench scale
phase (Li et al., 2017). Physical methods such as centrifugation and
filtration have elevated costs (DOE, 2016; Mata et al., 2010) whereas
sedimentation and flotation methods have low efficiencies without
previous flocculation (Barros et al., 2015; DOE, 2016). Chemical
methods, on the other hand, cause environmental impacts and biomass
contamination, limiting their applications (Wan et al., 2015).
The use of filamentous fungi for bioflocculation can be an effective
method for biomass harvesting, significantly reducing costs associated
with this step and allowing economically viable renewable energy
production (Bhattacharya et al., 2017; Chen et al., 2018; Choi et al.,
2016; Li et al., 2017; Prajapati et al., 2016). However, mechanisms of
this bioflocculation method are still uncertain (Bhattacharya et al.,
2017). Although separation of photosynthetic and fungal cells is not
possible, the pellets from coculture can be used as a concentrated bio-
mass to produce biogas (Prajapati et al., 2016), reducing anaerobic
digester volume, which lower costs.
In this study, the bioflocculation potential of Anabaena siamensis,
Anabaena variabilis, Spirulina maxima, and Synechococcus subsalsus with
Aspergillus niger pellets was assessed. The influence of factors such as
initial concentration of cyanobacterium, fungal:cyanobaterial ratio,
concentration of organic matter, and pH on bioflocculation efficiency of
S. subsalsus was evaluated. In addition, an attempt was made to es-
tablish a relationship between harvesting efficiency, pellet diameter,
and cyanobacterial biomass densification.
Bioresource Technology 294 (2019) 122167
Effects evaluated on bioflocculation
Cyanobacterium initial Fungal:cyanobacterial ratio Organic matter pH
concn. concentration
Synechococcus subsalsus
1:1 1:1 and 1:5 1:5 1:5
RFMb RFMb 14 to 96% sup.c 14% sup.c
without adjustmentd without adjustmentd without adjustmentd 3–8
0.345 and 0.738 0.345–0.729 0.623–0.729 0.793
24 h 24 h 24 h and 48 h 24 h
2. Materials and methods
2.1. Microorganisms, cyanobacteria suspensions, and pellet production
A. niger culture (Laboratory of Petroleum Microbiology, School of
Chemistry, Federal University of Rio de Janeiro) was maintained on
Potato Dextrose Agar (PDA) slants at 28 °C. Fungal spore suspension
(105 spores/L) was inoculated into 100 mL liquid medium (10 g/L su-
crose, 15 g/L yeast extract), in an orbital shaker, at 28 °C/150 rpm for
24 h for pellet production.
Cyanobacteria species A. siamensis, A. variabilis, and S. maxima were
supplied by the Laboratory of Applied Studies in Photosynthesis,
Institute of Chemistry, Federal University of Rio de Janeiro, and S.
subsalsus by the Microalgae Bank Aidar & Kutner, Oceanographic
Institute, University of São Paulo. For preliminary bioflocculation tests,
suspensions of each cyanobacteria were obtained in cultures in 250 mL
erlenmeyer flasks with 150 mL of BG-11 medium. The flasks were kept
at 26 °C, without agitation, under a 12:12 h light-dark cycle. In the light
period, lamps with a light intensity of 40 μmol/m2 s were used. For
bioflocculation tests conducted with S. subsalsus, cyanobacterium cells
were grown in 500 mL erlenmeyer flasks with 300 mL of BG-11 medium
at 23 °C/150 rpm for 30 days, under a 12:12 h light–dark cycle and light
intensity of 43 μmol/m2 s.
2.2. Preliminary bioflocculation tests
Preliminary bioflocculation tests (Table 1) were conducted by
adding pre-cultivated A. niger pellets into cyanobacteria suspensions at
1:1 fungal:cyanobacterial ratio (on dry weight basis). The 100 mL of
coculture was maintained in 500 mL erlenmeyer flasks incubated in
orbital shaker at 28 °C/100 rpm for 24 h. Bioflocculation efficiency (%)
was determined by the equation (1 − ODf/ODi) × 100), where ODi is
the initial optical density and ODf is final optical density, both at
750 nm (except for S. maxima, for which optical density was measured
at 730 nm). Tests were performed in duplicate.
2.3. Experiments with Synechococcus subsalsus
Bioflocculation experiments with S. subsalsus and A. niger pellets
were conducted under the same conditions described in Section 2.2,
with coculture times of 24 and 48 h. Bioflocculation efficiency was
determined by the same equation in Section 2.2, with OD at 750 nm.
The influence of factors such as the cyanobacterium initial con-
centration, fungal:cyanobacterial ratio, organic matter concentration,
and pH of coculture on bioflocculation efficiency was evaluated in
different experiments whose conditions are shown in Table 1. The effect
2
H.R. Oliveira, et al.
Table 2
Masses of sucrose and yeast extract added to the coculture and respective
supplementation percentages of the original fungal culture medium con-
centration.
Condition Sucrose (g) Yeast extract (g) Supplementation percentage (%)a
0 0 0 0
1 0.14 0.21 14
2 0.46 0.69 46
3 0.82 1.23 82
4 0.96 1.44 96
a
Original fungal culture medium contains 10 g/L sucrose, and 15 g/L yeast
extract.
of organic matter concentration in coculture was assessed by adding
fungal medium components to coculture, according to Table 2. In these
experiments, chemical oxygen demand (COD) of coculture supernatant
and pellet diameter were also evaluated. In the experiments evaluating
the pH effect of the coculture, the pH was adjusted with 1 mol/L H2SO4.
2.4. Analytical methods
Optical density (OD) was measured in Shimadzu UV-1800 or Hach
DR3900 spectrophotometers at wavelengths specified in Sections 2.2
and 2.3. Total suspended solids (TSS) concentration or dry weight of
cyanobacteria and fungus suspensions as well as the COD of coculture
supernatant were performed according to standard methods (Greenberg
et al., 2005).
3. Results and discussion
3.1. Preliminary bioflocculation tests
Results of bioflocculation tests of A. siamensis, A. variabilis, S.
maxima, and S. subsalsus with pre-cultivated A. niger pellets for 24 h
proved that bioflocculation efficiency depends on cyanobacteria spe-
cies.
A. siamensis and A. variabilis had final OD higher than initial OD.
Interaction of cyanobacteria cells and fungal hyphae was not enough to
reduce cyanobacterial concentration in the coculture. The increase in
OD was probably due to cyanobacteria growth from residual organic
matter from fungal medium, as both species are capable of hetero-
trophic growth (Khetkorn et al., 2010; Park et al., 2013). For S. maxima
and S. subsalsus, the supernatant was clearer in 24 h, reaching average
bioflocculation efficiencies of 77% and 94%, respectively.
The results obtained can be attributed to differences between the
species of cyanobacteria studied. All species bear a Gram-negative type
of cell wall, which includes a peptidoglycan layer and an outer mem-
brane outside of the cytoplasmic membrane (Hoiczyk and Hansel,
2000). Both species of the genus Anabaena are filamentous N2-fixing
heterocystous cyanobacteria (Khetkorn et al., 2010; Thiel et al., 2014).
The species S. maxima also has filamentous morphology, but does not
form heterocyst. The Synechococcus species is characterized by its co-
coid form and small cell size (Lee, 2008). Since S. subsalsus (coccus) and
S. maxima (filamentous) presented bioflocculation efficiencies with A.
niger pellets of 94% and 77%, respectively, cell morphology should not
be a determining factor of the bioflocculation mechanism between
these species.
The aggregation of microalgae and fungi can also be due to cell
surface charges. Most microalgae have negative charges on their cell
surface due to the presence of carboxylic (–COOH), amine (–NH2) and
phosphate (–PO4) groups. At pH above 4–5, the carboxylic groups are
dissociated and the cells gain a negative charge. While at the highly
acidic pH of the fungal culture, the surface groups are protonated, re-
sulting a net positive charge on the fungal hyphae. The difference in
charges indicates the ability of fungal cells to collect microalgae (Zhang
3
Bioresource Technology 294 (2019) 122167
and Hu, 2012; Bhattacharya et al., 2017). However, at the pH of the
highest bioflocculation efficiency (pH around 8), both A. niger
(Zamalloa et al., 2017) and S. subsalsus (Hao et al., 2017) have probably
net negative charge and it should not be a factor causing attraction in
the aggregation process.
A striking feature that differentiates the studied genera is the pre-
sence of cellulose in the surface structures of the cells. While the genus
Anabaena is marked by the expressive presence of a mucilaginous
sheath containing cellulose (Nobles et al., 2001; Tien et al., 2005),
species of the other two genera do not have cellulose and have a more
discreet sheath (Kuhn and Winston, 2007; Muthulakshmi et al., 2012;
Nobles et al., 2001). A thick sheath containing cellulose can impair the
interaction of the surfaces of fungal cells and hyphae, which would
explain the low bioflocculation efficiencies obtained with both Ana-
baena species. Bioflocculation of algal biomass using Aspergillus niger
pellets has been shown to have good efficiencies with Chlorella vulgaris
(Gultom et al., 2014), which also does not have cellulose in its outer
structures (Gerken et al., 2013).
Species of the genera Synechococcus and Anabaena have already
been identified in lichens (Kumar et al., 2013). In lichen formation,
fungi cells can identify compatible microalgae from affinity with sur-
face glycoproteins (Diaz et al., 2016). Moreover, surface proteins were
reported to play an important role in bioflocculation of microalgae with
Aspergilllus niger (Li et al., 2017). Therefore, a potential reason for the
differences found is the difference in cyanobacteria surface proteins.
The presence of pore-forming proteins may also be a factor in the in-
teraction between cyanobacterial cells and fungi, since the absence of
pores in Anabaena species has been reported, while the presence of
pore-forming proteins was noted in Synechococcus and Spirulina species
(Hansel and Tadros, 1998; Palinska and Krumbein, 2000).
Although the bioflocculation mechanism of A. niger and cyano-
bacterial cells was not the target of this study, based on the observed
differences, it can be summarized that the occurrence or not of bio-
flocculation depends on the cyanobacterial species evaluated.
Considering only the cyanobacterial species and the fungus used in this
study, there is evidence that the bioflocculation efficiency may be re-
lated to the absence of cellulose in the cell wall, the presence of pores
and a thin mucilaginous sheath. However, no studies were found that
relate the cellular structures and composition of photosynthetic mi-
croorganisms to the bioflocculation efficiency achieved with fila-
mentous fungi. Therefore, considering that the interaction of A. niger
pellets was more efficient with S. subsalsus, experiments evaluating the
effect of various coculture conditions were conducted only with this
species.
3.2. Experiments with Synechococcus subsalsus
3.2.1. Effect of cyanobacterium initial concentration
Experiments with cyanobacteria suspensions from larger and
shorter cultures were conducted to evaluate the effect of cyano-
bacterium initial concentration on bioflocculation efficiency. More
concentrated biomass (0.738 g TSS/L) resulted in much lower bio-
flocculation efficiency (19%). Gultom et al. (2014) also found that
when the initial algal biomass concentration was higher, fungal growth
was impaired and bioflocculation occurred to a lesser extent. While
almost all less concentrated biomass (0.345 g TSS/L) was attached to
fungal pellets. In this condition the pellets became larger and com-
pletely green, while the coculture medium was practically without
suspended cyanobacteria, reaching bioflocculation efficiency of 94%.
One explanation would be the culture age and phase. Microalgae
grown for a longer time and, therefore, with a higher cell age, excrete
larger amount of dissolved organic matter into the medium, often re-
ferred to as algal organic matter (AOM). This organic matter is com-
posed of neutral and charged polysaccharides, proteins, nucleic acids,
lipids, and small molecules, and has been shown to differ between
species and cultivation conditions. Mikulec et al. (2015) report that the
H.R. Oliveira, et al. Bioresource Technology 294 (2019) 122167

removal of AOM from the microalgae suspension increased the effi-

ciency of high molecular weight cationic polyacrylamide flocculant.
Vandamme et al. (2016) studied the influence of changes in cell prop-
erties and in the quality and composition of AOM using Chlorella vul-
garis as a model species. The authors found that the total carbohydrate
content (> 3 kDa) of organic matter fraction increased and inhibited
alkaline flocculation over time.
The best of our knowledge, no previous study has reported the re-
lationship between quantity and composition of AOM with flocculation
efficiency of cyanobacteria with fungal biomass. However, AOM, in
larger amounts in the older cell cultures, may have interacted with the
cells, reducing bioflocculation efficiency, as observed in chemical
flocculation.

3.2.2. Effect of fungal:cyanobacterial ratio Fig. 1. Bioflocculation efficiency of S. subsalsus with A. niger pellets, at fun-
When experiments with S. subsalsus were conducted maintaining the gal:cyanobacterial ratio of 1:5 (on dry weight basis), 28 °C/100 rpm for 24 h,
same conditions but reducing fungal:cyanobacterial ratio to 1:5 (on dry without and with different organic supplementation percentages (as shown in
weight basis), flocculation efficiency was 1.6 times lower than with 1:1 Table 2).
ratio, achieving only 59%.
A. niger pellets were used as a kind of bioflocculant. A lower fun-
slightly increased in the first 24 h (from 101 to 134 mg/L), probably
gal:cyanobacterial ratio (e.g. 1:5) means fewer pellets for the same
because the values were close to the detection limit of the method.
initial cyanobacterial concentration. Therefore, fewer pellets would
Considering the remaining conditions, the highest COD intake (35%)
have the same effect as lower flocculant dosage, reducing biofloccula-
occurred in Condition 1, followed by Conditions 2 (26%) and 4 (16%).
tion efficiency, as obtained in this study. This result is in accordance
Between 24 and 48 h, COD consumption was 42%, 54%, and 48% in
with other studies which found that the efficiency decreases with fewer
Conditions 1, 2, and 4, respectively. In Condition 1, the COD con-
number of pellets added to coculture (Bhattacharya et al., 2017; Chen
sumption was practically the same in 24 and 48 h of coculture. How-
et al., 2018; Choi et al., 2016).
ever, under conditions with more supplementation (Conditions 2 and
According to Choi et al. (2016), for the same initial concentration of
4), the COD consumption was higher between 24 and 48 h, indicating a
cyanobacteria, the fewer pellets applied, the more cyanobacteria cells
faster consumption of organic matter in this period.
attached to each single pellet, yielding the larger pellets. In this study,
This variation may indicate a relationship between the consumption
larger pellets were also observed at the end of coculture with lower
of organic matter and aggregation of cyanobacteria. Evaluating the
fungal:cyanobacterial ratio.
difference in bioflocculation efficiencies in 24 and 48 h (shown in
As the pellets used were collected directly from A. niger culture
Fig. 1) for the different supplementation conditions, the greatest var-
without any washing step, residual culture medium containing soluble
iations in bioflocculation efficiencies occurred in periods of faster COD
organic matter not used in the growth and pelletization of the fungus
consumption. Under the conditions with more supplementation (Con-
was also transferred to coculture. By lowering fungal:cyanobacterial
ditions 2 and 4), in which bioflocculation efficiency was low after 24 h
ratio, less pellets were added to coculture, less residual culture medium
and elevated after 48 h, COD consumption was faster (2 to 3 times) after
and less organic matter were removed from the fungal culture for co-
the first 24 h of coculture. On the other hand, in the condition with less
culture. Thus, the reduced organic matter might have influenced the
supplementation (Condition 1), bioflocculation efficiency was already
reduced efficiency. Studies suggest that lower carbon concentration or
relatively high after 24 h, increasing slightly after 48 h. In this condi-
its absence in coculture impairs flocculation with premade pellets
tion, the same COD consumption was observed after 24 and 48 h.
(Chen et al., 2018; Choi et al., 2016). In this study, the results obtained
Pellet diameter in cocultures increased in all supplementation con-
with different concentrations of organic matter in coculture are shown
ditions evaluated (Table 3). The largest size was achieved in the highest
in Section 3.2.3 below.
supplementation condition after 48 h. In the condition without sup-
plementation (Condition 0), there was almost no increase in pellet size,
3.2.3. Effect of organic matter
but the green color assumed by the pellets during coculture was visible.
To evaluate if the concentration of organic matter would affect the
In the remaining conditions, pellet size considerably increased. In
bioflocculation of S. subsalsus with A. niger, different proportions of
Conditions 2 and 4, the increase was linear during coculture. However,
yeast extract and sucrose in the coculture were studied. Experiments
in Condition 1, pellet increase was less between 24 and 48 h.
were conducted with a fungal:cyanobacterial ratio of 1:5 (on dry weight
In Fig. 2, COD consumption and pellet diameter data in the two
basis), a condition in which a smaller volume of the fungus residual
evaluated periods (0 to 24 h and 24 to 48 h) were plotted with the
culture medium was added to the coculture, and the results are in
bioflocculation efficiency, separating the data in which the efficiency
Fig. 1.
was low (up to 60%) from data in which efficiency was high (> 60%).
Only the lowest supplementation (Condition 1, Table 2) achieved
The trend lines in Fig. 2A and B indicate that both COD consumption
higher bioflocculation efficiency (70% with 24 h and 84% in 48 h) than
and pellet diameter are inversely proportional to the efficiency when
obtained without supplementation in 24 h (< 60%). However, after
bioflocculation efficiency is low. That is, both are more related to
48 h of coculture, all conditions with supplementation exhibited greater
fungus growth than to cyanobacteria aggregation to fungal pellets. On
efficiencies, achieving 98% (Conditions 3 and 4, Table 2), while the
the other hand, with high bioflocculation efficiencies, there is a direct
condition without supplementation (Condition 0, Table 2) remained at
relationship between efficiency and COD consumption and pellet dia-
58% efficiency. These results seem to indicate that the fungus uses re-
meter.
sidual organic matter of the culture medium for growth and increase of
The increase of bioflocculation efficiency and faster pellet formation
the pellets in the first 24 h, and later aggregation of cyanobacteria oc-
in coculture has been reported when higher carbon concentrations are
curs.
used (Chen et al., 2018; Gultom et al., 2014; Zhou et al., 2013). Choi
Table 3 lists the COD reduction and pellet growth of coculture su-
et al. (2016), who used pre-cultivated Aspergillus oryzae pellets to assist
pernatant. The COD decreased in all supplementation conditions eval-
Synechocystis sp. flocculation, reported the addition of carbon sources to
uated. However, in the condition without supplementation, COD

4
H.R. Oliveira, et al. Bioresource Technology 294 (2019) 122167

Table 3
Supernatant COD and pellet diameter during coculture of S. subsalsus with A. niger, at fungal:cyanobacterial ratio 1:5 (on dry weight basis), 28 °C/100 rpm, and
different carbon supplementations.
Time of coculture (h) Condition/ Carbon supplementation

Condition 0 Condition 1 Condition 2 Condition 4

0% 14% 46% 96%

a
COD (mg/L) ∅b (mm) COD (mg/L) ∅ (mm) COD (mg/L) ∅ (mm) COD (mg/L) ∅ (mm)

0 101 2.0 2428 2.0 6625 2.0 16,450 2.0

24 134 2.5 1587 4.5 4898 4.0 13,793 5.5
48 95 2.5 914 5.5 2262 6.0 7180 9.0

a
Mean chemical oxygen demand of coculture supernatant.
b
Mean pellets diameter.

the initial period of the coculture. This factor may also have been the
reason why at the beginning of the coculture higher supplementation
conditions reached lower efficiency than the conditions with less or-
ganic matter.

Fig. 2. Relationship of bioflocculation efficiency with COD consumption in 24 h
(A) and pellets diameter (B) in coculture of S. subsalsus and A. niger with fun-
gal:cyanobacterial ratio of 1:5 (on dry weight basis), 28 °C/100 rpm, with and
without supplementation: efficiencies lower than 60% (○) and efficiencies
higher than 60% (●).
obtain high bioflocculation efficiencies, as verified in the present study.
Furthermore, studying the addition of alternative carbon sources to
glucose (xylose, glycerol, and sodium acetate), researchers found that
the initial bioflocculation efficiency (1 day of coculture) was higher
with alternative sources, whereas after 2 days of coculture, the ex-
periment in which glucose was used as carbon source achieved higher
efficiencies. According to the authors, there would be a greater het-
erotrophic growth of the cyanobacteria in the medium with glucose in
3.2.4. Effect of pH
A bioflocculation test was performed with the best result in 24 h
(Condition 1) at different pH values, in order to evaluate the effect of
this variable on bioflocculation efficiency. For all studied conditions,
the appearance of coculture pellets were similar to the obtained in
Condition 1 without pH adjustment, except for pH 3, in which pellets
did not grow and the supernatant acquired a yellowish color.
Intermediate pH values (4 and 5) resulted in intermediate bio-
flocculation efficiencies (41% and 38%, respectively), while the con-
dition that reached the highest efficiency was at pH 6 (61%). The
control (without pH adjustment), in which initial pH was 8.1, reached a
bioflocculation efficiency lower than all other studied conditions
(17%), except that of pH 3, in which the efficiency could not be cal-
culated due to the increase in OD.
In previous studies, higher pH values contributed to a higher per-
centage of Chlorella vulgaris biomass in cocultured A. niger pellets due to
the favoring of microalgae growth at more alkaline pH (Zamalloa et al.,
2017), which would explain the higher bioflocculation obtained in this
study at pH 6 compared at pH 4 and 5. Additionally, pH 6 is closer to
the pH of coculture without adjustment (8.1), which required less
H2SO4 addition. The addition of larger amounts of acid may have af-
fected the aggregation mechanism of cyanobacteria cells, decreasing
the efficiency. However, these results differ from other studies. Chen
et al. (2018) reported efficiencies above 80% even at low pH values (3,
3.5, and 4) in the bioflocculation of Chlorella sp. with Penicillium sp.
Studies in higher pH ranges (5.4–7.4) also did not indicate the influence
of pH on the bioflocculation efficiency of Synechocystis sp. with Asper-
gillus oryzae (Choi et al., 2016). However, these studies have evaluated
a more limited pH range. Probably a wider range of pH values could
result in quite different bioflocculation efficiencies. Li et al. (2017)
reported that highly acidic and alkaline conditions inactivated the
flocculation activity of mycelial pellets and achieved higher bio-
flocculation efficiencies in alkaline pH (8–9). This could be explained
due to the significant impact that changes in pH may have on the
surface properties of the cells and the associated microalgae attachment
or entrapment (Li et al., 2017). In addition, optimal pH reported was
different in each study performed. This indicates that the conditions to
achieve higher efficiencies may vary when different species are applied
in the bioflocculation process.
3.2.5. Densification analysis
In the condition that achieved the highest bioflocculation efficiency
in 24 h (fungal:cyanobacterial ratio of 1:1, at 28 °C/100 rpm, without
supplementation), the cyanobacteria suspension was concentrated 10.3
times, from 0.216 g/L in the suspension to 2.236 g/L in the fungal

5
H.R. Oliveira, et al.
Fig. 3. Relationship of concentration factors of S. subsalsus with A. niger with
different pellets diameters (A) and bioflocculation efficiencies (B).
pellets. On the other hand, in the condition that had the highest bio-
flocculation in 48 h (fungal:cyanobacterial ratio of 1:5, at 28 °C/
100 rpm, and with 96% supplementation), the concentration factor was
only 3.2, from 0.907 g/L in the suspension to 2.904 g/L in the fungal
pellets. The condition with the highest concentration factor was with
fungal:cyanobacterial ratio of 1:5 and without supplementation, at
28 °C/100 rpm, which achieved a flocculation efficiency of only 59%,
but concentrated cyanobacteria biomass 62.8 times, from 0.9 to 56.5 g/
L.
Although the condition with 96% supplementation achieved a
higher bioflocculation efficiency, the concentration of cyanobacteria in
the pellets was lower, because pellet size was larger in this condition,
making their volume also larger. As the growth of cyanobacteria during
coculture was not quantified, there is the possibility of a larger mass of
cyanobacteria in the pellets than only that removed from the super-
natant.
Zamalloa et al. (2017) studied the bioflocculation of Chlorella vul-
garis with Aspergillus niger and also observed that when the pellet dia-
meter was smaller (2.6 ± 0.2 mm at pH 9), the concentration of mi-
croalgae cells in the pellets was higher. The authors attribute this result
to greater light penetration and better mass transfer in smaller pellets.
When compared to other biomass densification methods, the con-
centration obtained was lower than that achieved by more traditional
methods. Algae biomass can be concentrated up to 10 times with flo-
tation (Garg et al., 2012), with flocculation with chitosan up to 44
times, and with chemical flocculation with FeCl3, up to 50 times (Lama
et al., 2016). With centrifugation, much higher concentration factors of
up to 150 can be achieved (Grima et al., 2003). However, all these
methods involve the addition of chemicals (except for centrifugation,
which involves high energy costs), which contaminate the biomass,
limiting its posterior application.
Fig. 3 compares concentration factors of S. subsalsus with A. niger
6
Bioresource Technology 294 (2019) 122167
with different diameters and bioflocculation efficiencies. It indicates
that smaller pellets would be much more efficient for densification,
since concentration factors depend more on the final pellet diameter
than on flocculation efficiency itself. Therefore, priority should be given
to coculture conditions that result in high bioflocculation efficiencies
with lower pellet growth.
4. Conclusions
Among the four species of cyanobacteria evaluated, S. subsalsus
presented the highest bioflocculation efficiencies with A. niger pellets
(up to 98%). The factors that contributed to higher bioflocculation ef-
ficiencies of S. subsalsus in the coculture with the fungus include lower
initial S. subsalsus concentration, higher fungal:cyanobacterial ratio,
more organic matter in coculture (supplementation), and pH 6. The
highest concentration factor (62.3) was obtained with smaller pellets,
indicating the need to search for coculture conditions that lead to high
efficiencies with lower pellet growth.
Acknowledgments
This work was supported by grants from the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq – grant number
303406/2017-8), Fundação de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ – grant number E-26/201.327/2014), and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil
(CAPES – Finance Code 001). The authors also thank the Laboratório de
Hidrorrefino, Engenharia de Processos e Termodinâmica Aplicada, at
Escola de Química (UFRJ) for providing cultures used in the pre-
liminary bioflocculation tests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2019.122167.
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