Journal of Water Process Engineering 43 (2021) 102259

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Journal of Water Process Engineering

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Effect of hydrodynamics on autoflocculation and gravity sedimentation of

Chlorella vulgaris
Lin Li a, b, Wenhong Chen a, b, Yuyu Wang a, b, Yao Zhang a, b, Huaimin Chen c, *
a
Key Laboratory of Poyang Lake Wetland and Watershed Research, Ministry of Education, Jiangxi Normal University, Nanchang 330022, Jiangxi, China
b
School of Geography and Environment, Jiangxi Normal University, Nanchang 330022, Jiangxi, China
c
School of Materials Engineering (School of Environmental Engineering), Changzhou Institute of Industry Technology, Changzhou 213164, China

A R T I C L E I N F O A B S T R A C T

Keywords: Autoflocculation followed by gravity sedimentation is a promising method for harvesting of microalgae. How­
Flocculation ever, the effect of hydrodynamics on spontaneous flocculation of Chlorella vulgaris is yet uncertain. In this study,
Chlorella vulgaris where quantitative analysis was conducted to study the effect of hydrodynamics on autoflocculation of Chlorella
Hydrodynamics
vulgaris, it was found that hydrodynamic turbulence enhanced autoflocculation efficiency by 40–53.3%. Auto­
Harvesting
flocculation efficiency increased first and then decreased with the increase of turbulence strength. With shear
strength of 0.0069 N/m2 and energy dissipation efficient ε of 0.45 × 10− 3 m2/s3, the average floc size and
settling velocity achieved their maximum at 373.5 ± 36.4 μm and 2.17 ± 0.29 m/h respectively. However, after
shear strength exceeded 0.0115 Nm− 2 and energy dissipation efficient exceeded 1.25 × 10− 3 m2/s3, the Chlorella
vulgaris flocs started to disaggregate under the shearing effect. Hydrodynamic turbulence not only increased the
probability of contact between Chlorella vulgaris cells, but also the enhanced the accumulation of EPS, which
serves as bridge for autoflocculation after binding with the bivalent cation presented in the aqueous medium.
Under hydrodynamic conditions, Chlorella vulgaris flocs start to settle by gravity, with separation efficiency
reaching up to 94.5 ± 4.5%. Therefore, hydrodynamic control presents a new approach for low cost harvesting of
algae products.

1. Introduction products are mainly harvested by centrifugation, which involves high

Under the context of global resources scarcity, development of clean
and renewable recourses from microalgae biomass is of broad prospec­
tive and significance. Mass production of microalgae, which requires no
arable lands and thus avoiding land competition with other traditional
plants, has not been widely practiced [1]. Currently, the major bottle­
neck for commercial utilization of microalgae as bio-energy is the har­
vesting of microalgae [2]. Separation of microalgae from aqueous
medium accounts for 20–30% of the total biomass utilization cost due to
their high water content, which is difficult to remove [3,4]. Because of
their small size and similar density to water, microalgae tend to form
suspension in aqueous medium with low biomass concentration (about
0.1–2 g dry weight/L) [3,5]. As a result, the settling velocity of micro­
algae in aqueous medium is lower than 10− 6 m/s.
Commonly used methods for harvesting of microalgae include floc­
culation, gravity sedimentation, centrifugation, filtration, ultra-
filtration and flocculation-flotation [6,7]. At present, microalgae
energy consumption. Low energy consumption concentrating process as
pretreatment prior to centrifugation can be used to significantly reduce
the overall energy consumption for harvesting of microalgae products
[8]. Membrane filtration is an alternative technically feasible. However,
it has such disadvantages as membrane pollution, high membrane
replacement cost, etc. In addition, membrane filtration performance is
also dependent on microalgae density [9,10]. Chemical induced floc­
culation and gravity concentrating, as a promising method to reduce
microalgae harvesting cost and energy consumption [11], involves
addition of chemical flocculants, which not only increases operating cost
but also leads to contamination of microalgae products. In this study, a
flocculation process without addition of chemical flocculants was
investigated to realize preliminary concentrating and separation of
microalgae by gravity sendimentation [12].
According to previous studies, autoflocculation occurs when envi­
ronmental conditions are changed. Microalgae was found to flocculate
spontaneously and settle by gravity with the presence of bacteria

* Corresponding author.
E-mail address: huaiminchen@163.com (H. Chen).

https://doi.org/10.1016/j.jwpe.2021.102259
Received 17 March 2021; Received in revised form 4 August 2021; Accepted 6 August 2021
Available online 18 August 2021
2214-7144/© 2021 Elsevier Ltd. All rights reserved.
L. Li et al. Journal of Water Process Engineering 43 (2021) 102259

[13,14]. Autoflocculation and gravity sedimentation of microalgae were

observed as a result of increased excretion of extracellular poly­
saccharides (EPS) under nitrogen and phosphate limiting conditions
[15,16]. However, the EPS are negatively charged, which would prevent
the microalgae from colliding with each other and forming flocculation
because of the electrostatic repulsion [17]. Some studies have shown
that the cation (such as Ca2+ and Mg2+) will reacted with the EPS,
forming bridge which allows for improved floc size of microalgae
[18,19]. Hydrodynamics could increase the frequency of algal collisions.
In examining the impact of hydrodynamic on microalgae growth,
Hondzo and Lyn observed autoflocculation in Scenedesmus quadricauda
[20]. Microalgae autoflocculation was detected when Leupold et al.
investigated the impact of turbulence on chlorella photosynthesis [21].
Under turbulent conditions, Gutiérrez et al. [22] conducted experiments
to investigate the effect of flocculants on flocculation of microalgae used
in urban sewage treatment, where flocculation of microalgae occurred.
However, the effect of hydrodynamics on flocculation characteristics of
microalgae has remained unclear. Fig. 1. Experimental Setup (unit: mm).
This study of experiments were conducted in annular tanks made of
PMMA (polymethyl methacrylate). The effect of hydrodynamic on
beyond this flow rate. Hence, the flow rate of the water stream, which
autoflocculation of microalgae was quantified under various flow re­
was controlled by varying speed motor, was set at 0 (control group,
gimes with different flow rates. The floc characteristics was quantified in
running at 5 cm/s, 3 times/day, 3 min per time), 5, 10, 15, 25 and
terms of floc size, average settling velocity, and flocculation efficiency,
35 cm/s (experimental group, continuous operation) respectively. The
etc., to evaluate the potential of hydrodynamics in harvesting of
experimental setup was sterilized in advance with UV light of 30 W for
microalgae for mass production.
2–4 h prior to experiments, which were conducted in illuminated
incubator, with temperature of 29 ± 0.5 ◦ C, illumination intensity of
2. Materials and methods 45 μmol photons/(m2 s), and light-dark cycle of 14:10 h.
On day 5 after inoculation, when the mixture began to exhibit
2.1. Algal strain and medium evident autoflocculation characteristics, the algae samples were
collected to measure floc diameter, settling velocity, extracellular
Chlorella vulgaris has been used in various sewage treatment studies polysaccharides (EPS) and flocculation efficiency.
due to its high pollution resistance and high treatment rate [23,24]. In
the study, the unicellular Chlorella vulgaris strain FACHB-30, which was 2.2.1. Floc diameter
obtained from Institute of Hydrobiology, Chinese Academy of Science, During sedimentation, Sony Camera (SONY FDR-AX30, Japan) was
were axenically transferred into a 250 mL conical flasks, followed by used to take images, which were used by image processing software to
addition of 100 mL aseptic BG11 culture medium [25]. The conical measure the settling velocity and diameter of Chlorella vulgaris flocs
flasks were cultivated in illuminated incubator, at temperature of under various hydrodynamic conditions [29]. The long axis a and short
29 ± 0.5 ◦ C, illumination intensity of 40 μmol photons/(m2 s), and light- axis b, which represents the length of the longest axis and the length of
dark cycle of 14:10 h. The flasks were shaken manually three times a the shortest axis of a floc respectively, were measured using the image
day. Under these conditions, the Chlorella vulgaris existed as unicellular processing software. The floc diameter was calculated based on equation
morphs and collected for use at exponential growth phase. √̅̅̅̅̅
d= ab. More than 300 flocs per sample were measured to determine the
In order to observe the self-flocculation effect of Chlorella vulgaris in
average diameter.
the process of advanced treatment of effluent, the aqueous medium used
was the effluent collected from the outfall of a sewage treatment plant in
2.2.2. Settling velocity
Nanchang, Jiangxi province, China and without any pretreatment before
During gravity sedimentation experiments, camera was used to re­
use. The heavy metal content was determined using ICP-AES method
cord the sedimentation path of Chlorella vulgaris flocs inside the PMMA
(iCAP-7400, Thermo, USA). TN, TP and CODMn were determined using
cylinder (diameter: 10 cm, height: 50 cm). Chlorella vulgaris flocs were
standard methods [26]. The content of water samples were shown in
collected and transferred gently using pipette into the sedimentation
Table 1.
cylinder, where effluent water was used as sedimentation medium. The
settling velocity was calculated based on modified Stoke’s equation
2.2. Experimental setup [30]. The number of Chlorella vulgaris flocs used for statistics was 300.

Strain was batch-cultured in annular tanks made of PMMA as shown
in Fig. 1. Driven by varying speed motor, the impeller blade forced the
water stream to move around the tank. Our previous study showed that
the growth of Microcystis was inhibited at a flow rate of over 35 cm/s in
the same device [27]. Zhong et al. [28] also found that the Microcystis
had the largest colony size at a flow rate of 32 cm/s, and then decreased
2.2.3. The EPS content and zeta potential
To investigate the impact of EPS on sedimentation of Chlorella vul­
garis flocs under different hydrodynamic conditions, the EPS was
extracted by method of [31], and determined by Phenol‑sulfuric Acid
Method. Sample of the same volume was taken to measure the algae dry

Table 1
Effluents wastewater parameters unit (mg/L).
Item Ca K Mg Na Al Ba Cd Co Cr Cu

Content 34.4 11.86 7.412 58.04 ND 0.0166 ND ND ND ND

Item Mn Ni Pb V Zn Fe TN TP CODMn
Content 0.0159 0.0127 0.0178 0.0224 0.0099 ND 15.54 1.15 9.19

2
L. Li et al.
weight by drying method with 60 ◦ C. Finally, the EPS content (mg/g)
was normalised by the algae dry weight. Zeta potential of algal cells was
measured using Zeta potentiometric analyzer (Zeta Plus, Brookhaven
Instruments Corp., Holtsville, NY).
2.2.4. Flocculation efficiency
Flocculation efficiency (FE) was calculated based on Eq. (1) as shown
below [13]:
FE(%) = (1 − ODt /OD0 ) × 100
where OD0 and ODt refer to the absorbance measured at 680 nm of the
aqueous medium respectively at time zero and time t after the motor was
switched off.
2.3. Flow regime measurement and quantitative hydrodynamic analysis
Acoustics Doppler tachometer (16 MHz MicroADV) was employed
for flow regime measurement. Turbulent flow can be treated as a big
vortex, which is unstable and tends to break up into smaller and smaller
eddies till it is ultimately converted into heat energy by viscous stress.
This heat energy is dissipated equally in all directions. Statistically, there
is no energy variation along all direction [32]. Energy dissipation rate
(ε) is used to represent the conversion of kinetic energy into heat energy
[33]. Kolmogorov length refers to the length scale of the smallest eddies,
at which level the turbulent kinetic energy is dissipated into heat by
viscosity [34]. Energy dissipation rate and shear strength are key pa­
rameters in evaluating the hydrodynamic characteristics of fluid flow
[35,36]. E(ω), the power spectra, was obtained based on spectral anal­
ysis of flow velocity, and average E(ω) was obtained based on the cor­
relation between ω and E(ω). The energy dissipation rate was calculated
based on the Eq. (2) as below:
( )−
1/2
ε = E(ω)2/3 w5/2 α− 3/2
u′ 2
where ω is angular frequency; α is a constant with a value of approx. 0.8;
u′ 2 is the mean square of the horizontal velocity fluctuation.
Kolmogorov length, the scale of the smallest eddy in the turbulent
a b
e f
Fig. 2. Autoflocculation of Chlorella vulgaris under hydrodynamic conditions, Figure a, b, c, d, e, f and g respectively corresponds to flow rate of 0, 5, 10 15, 25, 35
and 25 cm/s (scale: 10 mm).
Journal of Water Process Engineering 43 (2021) 102259
regime, was calculated based on Eq. (3) as below:
( / )1/4
ηk = ν3 ε (3)
Shear strength was calculated based on Eq. (4) as below:
τ = μγ (4)
Kinematic viscosity was calculated based on Eq. (5) as below:
μ = ρν (5)
(1)
Rate of strain was calculated based on Eq. (6):
γ = (ε/ν)1/2 (6)
where ρ is density of the liquid, which is approx. 1 mg/mL; ν is viscosity,
which is approx. 10− 6 m2/s.
3. Results and discussion
3.1. Images of Chlorella vulgaris autoflocculation regime
Under experimental conditions, autoflocculation occurred in both
control group and experimental group on day 1 after inoculation, but the
experimental group exhibited more evident autoflocculation charac­
teristics. The floc size increased with cultivation time. Fig. 2 presented
the images recorded during autoflocculation of Chlorella vulgaris on day
5 after inoculation. As shown in image a, b, c, e, f and g, which corre­
spond Chlorella vulgaris flocculation regime at flow rate of 0, 5, 10, 15,
25 and 35 cm/s respectively. After allowing the same sedimentation
cylinder corresponding to image e to settle for additional 16 min, as
illustrated in image g, the Chlorella vulgaris flocs settled to the bottom by
gravity with desirable algae and water separation performance. This
suggested that hydrodynamically induced autoflocculation can be a
promising approach for harvesting of Chlorella vulgaris.
(2)
3.2. The effect of hydrodynamics on autoflocculation of Chlorella vulgaris
After 5 days’ cultivation, the autoflocculation characteristics of
Chlorella vulgaris were analyzed as shown in Fig. 3. In the control group
c d
g
3
L. Li et al. Journal of Water Process Engineering 43 (2021) 102259

500 than the scale of the smallest eddy (ηKolmogrov is 387 μm) (Table 2). And
when shear strength above 0.115 × 0.1 N/m2, the flocs started to
disaggregate (Fig. 3). As shown in Fig. 4, the ζ -potential of the aqueous
400 medium in experimental group varied from − 10 mV to -5 mV, which is
Average floc diameter (μm)

closer to 0 mV than that of control group. The flocs formed in experi­

300
200
100
0 Fig. 5 that the autoflocculation efficiency under hydrodynamic effect
0 5 10 15
Flow rate (cm/s)
Fig. 3. Average floc size of Chlorella vulgaris under different hydrody­
namic conditions.
(static conditions), the standard floc diameter was 108 ± 9 μm. In the
experimental group with flow rate from 5 to 35 cm/s, the standard floc
diameter increased in turn from 230 ± 28 μm to 373 ± 46 μm and then
decreased to 227 ± 26 μm, with the maximum floc size occurring at flow
rate of 15 cm/s and ε of 0.45 × 10− 4 m2/s3. The floc size in the exper­
imental group and control group demonstrated significant difference
(p<0.01), suggesting fluid flow significantly promotes autoflocculation
of Chlorella vulgaris. As shown in Fig. 4, hydrodynamic conditions
considerably increased EPS content of Chlorella vulgaris cells (p < 0.01),
with the maximum EPS content reaching up to 142 ± 17 mg/g dry
weight at flow rate of 15 cm/s. The EPS with negative charge will
reacted with the cation present in the aqueous medium (such as Ca, Mg)
[19,37], forming bridge which allows for improved autoflocculation of
Chlorella vulgaris. In laboratory experiments under static conditions with
calcium ion concentration of 15 mg/L and 100 mg/L, Wang et al. [38]
observed that microalgae floc diameter reached 55 μm and 105 μm
respectively, which are smaller than the floc size observed in the
experimental group in this study. This may be caused by fluid flow,
which increased the contact probability of algae cells. In addition,
appropriate shear strength, by inhibiting the release of daughter cells
from the mother cells, also promoted spontaneous flocculation of algae
cells. Fluid flow, in turn, limits the floc size [39]. The floc size of chlorella
vulgaris at 15 cm/s flow rate was 373 ± 46 μm (Fig. 3), which is smaller
200
Extracellular polysaccharides
ζ-potential
Extracellular polysaccharides (mg/g)
mental group, though more stable than those formed in control group,
were still less stable compared with those generated under ideal floc­
culation conditions, where the ζ-potential of the aqueous medium equals
to 0 mV [40].
3.3. Analysis of hydrodynamic effect on autoflocculation and
sedimentation of microalgae
Autoflocculation characteristics of Chlorella vulgaris in aqueous me­
dium was analyzed as shown in Fig. 5 and Fig. 6. It can be seen from
25 35 ranged from 84 ± 3.1% to 95 ± 5%, considerably higher than that of the
control group under static conditions, which was (62 ± 3%) (p<0.01).
The autoflocculation efficiency was highest at flow rate of 15 cm/s.
As an important parameter in evaluating flocculation efficiency, the
settling velocity of Chlorella vulgaris flocs varies depending on floc size,
structure and density [41]. The average autoflocculation settling ve­
locity for control group and experimental group was 0.515 ± 0.05 m/h
and 1.47–2.17 m/h respectively, with the highest settling velocity
occurring at flow rate of 15 cm/s. The significant increase in settling
velocity under hydrodynamic conditions may be contributable to the
increase in microalgae density as a result of increased EPS accumulation.
With low investment and energy consumption [42], autoflocculation
and gravity settling can be used for harvesting of algae from the high
rate algal pond, which is used for domestic wastewater [43]. Based on
PNK (Ponder-Nakamura-Kuroda) theory, the clean water production
efficiency Qc is associated with projected settling area (on the horizontal
plane) and algae settling velocity of the treatment work as shown in Eq.
(7) [44,45]:
( )
L
Qc = NWbv sinθ + cosθ (7)
b
where N, b and W are the number, spacing, and width of the plates,
respectively; v is the flocculation settling velocity, which is 2 m/h; L is
the length of the plate, which is 1 m; b is the width of the plate, which is
2 cm; θ is the included angle between the horizontal line and the object
to be settled, which is approx. 65.
If an open algae pond is used to treat wastewater with capacity of
80,000m3 per hectare per year, which is equivalent to 300m3 per day,
the plate separator required for harvesting of algae is less than 1 m2.
However, whether Chlorella Vulgari could further in-depth treatment of
the effluent of the sewage treatment plant needs to be further explored
0 in future studies.

4. Conclusions
160 -10

Based on hydrodynamic quantitative analysis, hydrodynamic

ζ-potential (mV)

120 -20
Table 2
Fluid mechanical parameters as a function of rotation rates in an annular PMMA
80 -30 tank.
Rotate Flow Energy Kolmogorov Viscous Rate of
speed rate dissipation rate scale stress strain
40 -40
(rpm) (m/s) ε × 10− 4 (m2/ ηK × 10− 3 (m) τ × 0.1 γ (s− 1)
s3) (N m− 2)

0 -50 0 – – – – –
0 5 10 15 25 35 8 0.05 0.05 0.671 0.023 0.576
Flow rate (cm/s) 24 0.10 0.20 0.474 0.046 1.153
40 0.15 0.45 0.387 0.069 1.729
72 0.25 1.25 0.300 0.115 2.882
Fig. 4. EPS content and ζ -potential value under different hydrody­
100 0.35 2.45 0.253 0.161 4.035
namic conditions.

4
L. Li et al. Journal of Water Process Engineering 43 (2021) 102259

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Declaration of competing interest
The authors report no declarations of interest.
Acknowledgements
This study was sponsored by the National Natural Science Founda­
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