bioengineering
Article
Dynamic Foam Characteristics during Cultivation of
Arthrospira platensis
Ameer Ali Kubar 1,2,† , Amjad Ali 3,† , Santosh Kumar 2 , Shuhao Huo 1, *, Muhammad Wajid Ullah 4 ,
Khulood Fahad Saud Alabbosh 5 , Muhammad Ikram 6 and Jun Cheng 2, *
Citation: Kubar, A.A.; Ali, A.; Kumar,
S.; Huo, S.; Ullah, M.W.; Alabbosh,
K.F.S.; Ikram, M.; Cheng, J. Dynamic
Foam Characteristics during
Cultivation of Arthrospira platensis.
Bioengineering 2022, 9, 257.
https://doi.org/10.3390/
bioengineering9060257
Academic Editors: Giorgos Markou
and Imene Chentir
Received: 8 May 2022
Accepted: 9 June 2022
Published: 16 June 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Bioengineering 2022, 9, 257. https://doi.org/10.3390/bioengineering9060257
1 School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China;
ameerali.kubar@yahoo.com
2 State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China;
santoshmalhi@zju.edu.cn
3 Research School of Polymeric Materials, School of Materials Science and Engineering, Jiangsu University,
Zhenjiang 212013, China; amjadali@zju.edu.cn
4 Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University,
Zhenjiang 212013, China; wajid_kundi@ujs.edu.cn
5 Department of Biology, College of Science, University of Hail, Hail 55476, Saudi Arabia; k.alabosh@uoh.edu.sa
6 Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad 22060, Pakistan; ikram@cuiatd.edu.pk
* Correspondence: huo@ujs.edu.cn (S.H.); juncheng@zju.edu.cn (J.C.)
† These authors contributed equally to this work.
Abstract: This study is aimed at understanding the serious foaming problems during microalgal
cultivation in industrial raceway ponds by studying the dynamic foam properties in Arthrospira
platensis cultivation. A. platensis was cultivated in a 4 L bowl bioreactor for 4 days, during which
the foam height above the algal solution increased from 0 to 30 mm with a bubble diameter of
1.8 mm, and biomass yield reached 1.5 g/L. The algal solution surface tension decreased from
55 to 45 mN/m, which favored the adsorption of microalgae on the bubble to generate more sta-
ble foams. This resulted in increased foam stability (FS) from 1 to 10 s, foam capacity (FC) from
0.3 to 1.2, foam expansion (FE) from 15 to 43, and foam maximum density (FMD) from 0.02 to 0.07.
These results show a decrease in CO2 flow rate and operation temperature when using the Foamscan
instrument, which minimized the foaming phenomenon in algal solutions to a significantly lower
and acceptable level.
Keywords: foam; stability; flow rate; photobioreactor; carbon dioxide; temperature
1. Introduction
Microalgae have been the subject of growing industrial research for years due to
their greater photosynthetic efficiency in utilizing CO2 , sunlight, and inorganic nutrients,
which in turn contribute to reducing the effects of global warming [1,2]. Many studies
have indicated that microalgae are an important source of high-value products and a
strong candidate for sustainable and eco-friendly energy sources worldwide; this brings
the potential to meet the need of rapidly growing economies, particularly in developing
countries, by offering fewer adverse atmospheric effects [3–7]. The microalgal raceway
ponds are usually 15 to 25 cm deep, supplied with a source of CO2, equipped with a paddle
wheel, and have guided barriers in the flow channel to ensure homogeneity. These raceway
ponds are generally lit by sunlight under controlled temperature and have a ready source
of water; this is currently the most industrially marketable technology because of its lower
costs compared to the closed bioreactors [8–13].
Foaming is desirable for a number of global industries, such as in the food indus-
try, where foam is of great importance for basic food textures owing to its lightness and
large specific surface area [14–16]. Several engineering processes, however, are directly
https://www.mdpi.com/journal/bioengineering
Bioengineering 2022, 9, x FOR PEER REVIEW 2 of 12

Bioengineering 2022, 9, 257 2 of 11

specific surface area [14–16]. Several engineering processes, however, are directly affected
by the unwanted
affected foams, foams,
by the unwanted such assuch in metal industries,
as in metal wastewater
industries, treatment
wastewater plants,
treatment and
plants,
anaerobic digesters. Foams can obstruct gas transport and render
and anaerobic digesters. Foams can obstruct gas transport and render the process ineffi- the process inefficient,
thus significantly
cient, increasing
thus significantly cost [17–20].
increasing FoamFoam
cost [17–20]. becomes problematic
becomes during
problematic algal algal
during culti-
vation when
cultivation it is formed
when to a level
it is formed to athat
levelcan
thathinder the regular
can hinder process process
the regular tasks. This phenom-
tasks. This
enon has remained
phenomenon unnoticed
has remained and been
unnoticed andignored for decades.
been ignored FoamsFoams
for decades. may last
mayfor short
last for
timestimes
short (up to(up
hours) on solution
to hours) on solutionsurfaces during
surfaces large-scale
during algal algal
large-scale cultivation in anin
cultivation open
an
pond, as shown in Figure 1. This results in adverse effects, such
open pond, as shown in Figure 1. This results in adverse effects, such as a decrease inas a decrease in growth,
loss of volume,
growth, lower light
loss of volume, lower penetration (since phototrophic
light penetration microalgal
(since phototrophic cultivation
microalgal strongly
cultivation
dependsdepends
strongly on light onenergy), pond structure
light energy), failure, failure,
pond structure and difficult manualmanual
and difficult cleaning of the re-
cleaning of
actor.
the Such Such
reactor. impacts can lead
impacts cantolead
economic losses losses
to economic of varying magnitudes.
of varying Numerous
magnitudes. studies
Numerous
have provided
studies advances
have provided in structural
advances engineering
in structural in reactor
engineering designdesign
in reactor for attaining maxi-
for attaining
maximum
mum algalalgal biomass
biomass yield;
yield; however,
however, thethe formationofoffoam
formation foamduring
during algal
algal cultivation in in
photo-bioreactors is still unreported in the existing literature
photo-bioreactors is still unreported in the existing literature [21–24]. [21–24].

Figure 1. Formation of stable CO2 bubbles at different foam heights.

Figure 1. Formation of stable CO2 bubbles at different foam heights.

Tounderstand
To understandthe theserious
serious foaming
foaming problems
problems that
that present
present during
during microalgal
microalgal cultiva-
cultivation
tion
in in industrial
industrial raceway
raceway ponds,ponds, the dynamic
the dynamic foamfoam properties
properties fromfrom A. platensis
A. platensis cultiva-
cultivation
tion were
were characterized
characterized in this
in this study
study to clarify
to clarify thethe processes
processes inin foamformation
foam formationandandfoam
foam
evolution. The reduced surface tension of algal cultures, combined with ionic
evolution. The reduced surface tension of algal cultures, combined with ionic sulfate and sulfate and
phosphate surfactant adsorption on bubble walls, was found to generate
phosphate surfactant adsorption on bubble walls, was found to generate more stable foams.more stable
foams.
This This resulted
resulted in an increase
in an increase in foaminstability,
foam stability, foam capacity,
foam capacity, foam expansion,
foam expansion, and
and foam
foam maximum
maximum density.density.

2.
2. Materials
Materials and
and Methods
Methods
2.1. Instrumental Setup
2.1. Instrumental Setup
The
Themain
main instrument
instrument used
used in
in this
this study
study was
was aa Foamscan
Foamscan system
system (Teclis
(Teclis Instruments,
Instruments,
Civrieux-d’Azergues,
Civrieux-d’Azergues, France). A schematic diagram of the Foamscan instrumentisisshown
France). A schematic diagram of the Foamscan instrument shown
in
in Figure 2. This
Figure 2. Thissystem
systemisisconnected
connected with
with software
software forfor precise
precise determination
determination of foam
of foam vol-
volume, liquid
ume, liquid fraction
fraction andandvolume,
volume,andandcontrolling
controllingthe
thegas
gas flow
flow rate,
rate, stirring
stirring speed,
speed, and
and
measuring the bubble size and distribution. Foamscan optically measures the foaming
measuring the bubble size and distribution. Foamscan optically measures the foaming ca-
capabilities by providing information on stability, drainage rate, and bubble size distribu-
pabilities by providing information on stability, drainage rate, and bubble size distribu-
tion [25]. Additionally, the foam column is layered with a temperature sensor inside the
tion [25]. Additionally, the foam column is layered with a temperature sensor inside the
tube. In the present study, the Foamscan system was used to determine the foam stability
tube. In the present study, the Foamscan system was used to determine the foam stability
(FS), foam capacity (FC), foam expansion (FE), and foam maximum density (FMD).
(FS), foam capacity (FC), foam expansion (FE), and foam maximum density (FMD).
Bioengineering 2022,
Bioengineering 9, 9,
2022, 257x FOR PEER REVIEW 3 3ofof12
11

Figure2.2.Schematic
Figure Schematicillustration
illustrationof
offoam
foam measurement
measurement system.
system.

2.2.
2.2.Foam
FoamGeneration
Generationand andStability
Stability Analysis
Analysis
For
Forfoam
foam generation,
generation, aa 50 50mL mLsample
samplesolution
solution waswas injected
injected through
through an inlet
an inlet into into
the
the foam column, equipped with a temperature sensor inside
foam column, equipped with a temperature sensor inside the tube. The experimental pa-the tube. The experimen-
tal parameters
rameters werewereset bysetusing
by using the connected
the connected software.
software. For allFor all experiments,
experiments, thefoam
the final final
foam
volume volume
limit limit
was setwasatset at 70The
70 mL. mL.foamThe was
foamgenerated
was generated by sparging
by sparging CO2 gasCO 2 gas
into theinto
in-
the injected
jected sample sample solution
solution in the in thecolumn,
foam foam column, and its volume
and its volume was controlled
was controlled with
with the help
the help
of the of the connected
connected software. software.
Gas sparging Gas sparging
was was automatically
automatically stopped oncestopped
the foamonce
volume the
foam
reachedvolume reached
the preset the
final preset
limit, andfinal
testslimit,
wereand tests were
performed performed
to separately to separately
investigate the ef-in-
vestigate
fects of COthe2 and
effects of CO2 and
temperature. Fortemperature.
CO2 flow rateFor CO
tests, the
2 flow rate
temperature tests,
wasthe temperature
fixed at 30 °C,
was fixed CO
at 30 ◦ C, whereas CO aeration was varied according to the testing parameters
whereas 2 aeration was varied 2 according to the testing parameters (200, 250, 300, 350,
(200, 250,mL/min).
and 400 300, 350,The andtemperature
400 mL/min). effectThe
wastemperature
determined at effect wastemperatures
varying determined at (20,vary-
25,
ing
30,temperatures
35, and 40 °C),(20, 25, 30,the
whereas 35,COand 40 ◦ C), rate
2 aeration whereas the CO
was fixed aeration
at 2300 mL/min. rateThewas fixed
foam sta-at
300 mL/min.
bility, The foam
also known as thestability,
half-life,also
was known as the in
determined half-life,
terms of was FE,determined
FC, and FMD, in terms
as de- of
FE, FC, and
scribed FMD, as described below:
below:
FEFEwas
wasdetermined
determinedas asthe
theratio
ratio ofof the
the total
total foam volume to the the liquid
liquid volume
volumewithin
within
the
thebubbles
bubblesafter
afterfoam
foamgeneration
generationisiscompleted
completed by by using
using Equation (1).

VfVf୤୭ୟ୫
FEFE=ൌ Vi foam (1)
(1)
Viliq୪୧୯− െVfVf
liq୪୧୯
where Vf୤୭ୟ୫ (mL) is the total foam volume after the completion of the foaming process,
Vi୪୧୯ (mL)
where is (mL)
Vffoam the liquid
is thevolume at the
total foam initial after
volume state,the Vf୪୧୯ (mL) is
andcompletion of the
the volume
foamingofprocess,
liquid
Viremaining
liq (mL) isafter
the liquid volume at
the completion of the
the initial
foaming state, and Vfliq (mL) is the volume of liquid
process.
remaining after
FC was the completion
determined of theoffoaming
as the ratio the totalprocess.
foam volume to the gas volume after foam
generation is completed by using Equation (2) [26].
Vf୤୭ୟ୫
FC ൌ (2)
Vf୥ୟୱ
Bioengineering 2022, 9, 257 4 of 11

FC was determined as the ratio of the total foam volume to the gas volume after foam
generation is completed by using Equation (2) [26].

Vffoam
FC = (2)
Vfgas

FMD was determined as the ratio of the difference between the initial liquid volume
and the final liquid volume with the final foam volume.

2.3. Surface Tension

The surface tension (mN/m) of the solution was measured by using a digital surface
pressure device via the du Noüy ring method. Briefly, the fluid samples were added
to a container with an automated adjustable height. After each reading, the ring was
thoroughly washed with double distilled water and then heated in a flame. Measurements
were performed three times for reproducibility and accuracy.

2.4. Cultivation Conditions

The A. platensis strain was cultivated with 4 L Zarrouk’s medium in a bowl photobiore-
actor aerated with the 15% CO2, and the flow rate was controlled at 300 mL/min using
a mass flow meter (Sevenstar CS200, Beijing, China). The experiment was conducted in
an artificial climate greenhouse with an indoor temperature of 30 ± 2 ◦ C, and the light
intensity was kept at 8000 ± 200 lux. Biomass was measured using optical density with
algal solution (10 mL) using a WFJ 7200 visible spectrophotometer at the wavelength of
560 nm with deionized water as blank. Error bars shown were found with Excel’s stan-
dard deviation (SD) function. The pH value throughout the study was determined using
an FE20 laboratory pH meter. The data for the dry weight curve of the A. platensis was
obtained by (y = 0.51x − 0.034, where y refers to dry weight, and x refers to OD). Each
measurement during all experiments was performed twice a day at 9:00 and 21:00 to ensure
reproductively and accuracy of the results, and each data throughout the figures is a mean
of three data with an error bar to represent uncertainty in the measurements [26].

2.5. Microscopic Observation

The bubble diameter of foam was determined, as described previously [22], by using a
Nikon inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) and calculated
by using software (NIS-Elements BR4.00.12). The bubble diameter was measured twice per
day for 4 consecutive days. Data were collected from at least 50 bubbles each time.

3. Results and Discussion

3.1. Foam Morphology during A. platensis Cultivation
The foam morphology above the solution was observed during algal cultivation. With
increasing microalgae density in solution, the surface tension decreased, which weakened
the interfacial properties at the air-liquid interface. Therefore, the microalgal solution
resulted in fast adsorption kinetics and high surface visco-elastic interface properties. These
properties are favorable for foam formation [15,27,28]. Figure 3 illustrates the process in-
volved in stable and unstable bubble formation and a typical mechanism of foam formation
during the cultivation of A. platensis. A sticky gel-like material was continuously excreted
through the walls of A. platensis, known as extracellular polymeric substance (EPS), that
generated an aggregate formation by adhesion between microalgae surfaces which were
trapped in the thin bubble films that promoted stable foam over the solution surface [27,28].
 Bioengineering
Bioengineering
Bioengineering 2022,
2022, 9, x FOR 9,9,257
2022,PEER REVIEW
x FOR PEER REVIEW 5 of 12 55 of
of 11
12

Figure 3. Figure
Mechanism
Figure3. of foam formation
3. Mechanism
Mechanism of foam
of during cultivation
foam formation
formation of A. platensis.
duringcultivation
during cultivation ofA.
of A.platensis.
platensis.

As shown Asinshown
As Figurein 4a,
in the foam
Figure
Figure height
4a,the
4a, the over
foam
foam the over
surface
height
height theof
over microalgae
the surface
surface ofsolution grad-solution
microalgae
of microalgae solution grad-
ually increased
gradually andincreased
reached
ually increased and the
andhighest
reached thevalue
reached the of
highest 30 mm
highest
value ofafter
value
30 mm 4 days
of 30after
mm (Figure
days4b).
4 after During
4(Figure
days (Figure 4b).
4b). During
this time,During
thetime,
this this
bubblethetime,
diameter
bubblethediameter
bubble
also diameter
increased andalso increased
reached
also increased and 1.8 mm
reachedand1.8reached
after 4 days
mm 1.8 4mm
daysafter
(Figure
after 4b). 4 days
(Figure 4b).
(Figure
Importantly, the4b). Importantly,
bubble diameter the bubble
close to the diameter
surface close
was to the
smaller surface
as was
compared smaller
Importantly, the bubble diameter close to the surface was smaller as compared to the bub- to the as compared
bub-
to
bles locatedthe
bles at bubbles located
the topatof
located topat
foams
the the
and
of top and
away
foams offrom
foamsthe
away and away
the from
solution.
from This the solution.
phenomenon
solution. This phenomenon
occurred
This phenomenon occurred
occurred
due to coarsening. due to
due to coarsening. coarsening.

Figure 4.Figure
(a) Foam
Figure 4.4.(a)formation
(a) Foam
Foam duringduring
formation
formation cultivation
during of A. of
platensis
cultivation
cultivation A. cultivation
A.ofplatensis
platensis in industrial
cultivation
cultivation raceway
in industrial
in industrial racewayraceway
ponds
ponds (b)(b)Qualitative
ponds and quantitative demonstration of increasing foam height and bubble diam-
Qualitative and quantitative demonstration of increasing foam height and bubble diameterdiam-
(b) Qualitative and quantitative demonstration of increasing foam height and bubble after
eter after eter
4 days of microalgal
after growth.
4 days of microalgal
4 days of microalgal growth. growth.

Bubbles Bubbles
generated
Bubbles by aeration
generated
generated by can resist
by aeration
aeration can
canbursting because because
resist bursting
resist bursting adsorption
because of the sticky
adsorption
adsorption of the
of the sticky
sticky
nature of algal
nature cells
natureofofalgal attached
algalcells
cells to bubbles
attached
attached led
to bubbles
to bubbles to the
led to formation
ledthetoformation of
the formationa resistant armor
of a resistant
of a resistant that
armor that armor that
slowed
slowed down
slowed
down the
the drainage,
down reduced reduced
the drainage,
drainage, reduced bubble breakage,
bubble breakage, and increased
bubble breakage,
and increased coarsening,
and increased
coarsening, whichresulted
coarsening,
which which
resultedinin more
more stable
resulted in more
stable and
and long-lasting
stable
long-lasting foam
foam[29].
and long-lasting AsAs
foam
[29]. the
[29].foam
theAsfoamheight
the foam rises,
height bubble
height
rises,rises, diam-
bubble bubble diam-
diameter
eter increases, andand
increases, bubbles
eter increases, and coarsen
bubbles bubbles from
coarsen
coarsen smaller
from frombubbles
smaller smaller
bubbles to larger
bubbles
to bubbles
to
larger larger
bubbles over time
bubbles
over due
over
time time
due to due
gas
to gas diffusion [30].
[30]. According
to gas diffusion
diffusion to the
[30]. According
According the Laplace–Young
Laplace–Young
to the Laplace–Young law,‘coarsening’
law, ‘coarsening’ involvesthe
law, ‘coarsening’
involves the
involves
transportthe
transportof of gas
transport between bubbles
of gasbubbles
gas between between duedue
bubblesto their
to theirdue differences in
to their differences
differences pressure,
in pressure,in leading
pressure,
leading to
to an an
leading in-
increase to in
anthein-
crease inaverage
the average
crease the bubble
inbubble diameter
diameter
average over
time time
overdiameter
bubble [31,32].[31,32].
over time [31,32].
 Bioengineering2022,
Bioengineering 2022,9,9,257
x FOR PEER REVIEW 66 of
of 11
12

3.2.Stability
3.2. StabilityofofFoam
Foamduring
duringBiomass
BiomassProduction
Production
Foamingwas
Foaming wasaagradual
gradualand andcontinuous
continuousprocess
processduring
duringthethe cultivation
cultivation ofof microalgae,
microalgae,
and 1.5 g/L
and g/Lofofmicroalgal
microalgal biomass
biomass waswasproduced
producedafterafter
4 days (Figure
4 days 5). When
(Figure 5). the biomass
When the
biomass
grew densergrewand denser and A.cells
A. platensis platensis
startedcells
to started
producetoEPS produce
aroundEPS around
their walls, their
a largewalls,
por-
ation
large
of portion
EPS wasofattached
EPS was to attached
the surface to of
theA.surface A. platensis
platensisofcells, whereascells,
somewhereas some
were released
were released
into the cultureinto the culture
medium. medium. The
The separation separation
of EPS from theofcell EPS from is
surface thea well-established
cell surface is a
well-established phenomenon
phenomenon [30,33,34]. [30,33,34].
The released EPS The released
formed EPSthin
stable formed
filmsstable
in thethin filmsand
solution in the
re-
solution and resulted in the formation of stable foams. This polymeric
sulted in the formation of stable foams. This polymeric substance, i.e., EPS, consists substance, i.e., EPS,
of
consists
sulfatedofsubstitutes
sulfated substitutes
(0.5–22%),(0.5–22%),
uronic acidsuronic acids (14–40%),
(14–40%), and polysaccharides.
and polysaccharides. The
The results
results
showed showed an association
an association between between the microalgal
the microalgal biomass biomass and surface
and surface tension.tension. The
The surface
surface
tensiontension continuously
continuously decreased
decreased with increasing
with increasing microalgal
microalgal biomass
biomass (Figure
(Figure 5). The
5). The am-
amphiphilic nature of the algal solution is due to the presence of uronic
phiphilic nature of the algal solution is due to the presence of uronic acids and peptides acids and peptides
in
inEPS
EPS[35].
[35].Therefore,
Therefore, thethe
emulsifying
emulsifying property of theofsolution
property was significantly
the solution enhanced,
was significantly en-
which is caused by the presence of rhamnose and fucose deoxy sugars
hanced, which is caused by the presence of rhamnose and fucose deoxy sugars [29,33,36]. [29,33,36]. During
the cultivation
During time, thetime,
the cultivation pH of thethe
pHsolution also increased,
of the solution although
also increased, only slightly,
although from
only slightly,
~9.8
from to~9.8
~10.2to(Figure 5). This5).slight
~10.2 (Figure This increase in pH during
slight increase foam formation
in pH during could becould
foam formation due tobe
the CO uptake.
due to the CO2 uptake.
2

Figure5.5.Formation
Figure Formationofofmicroalgal
microalgalbiomass
biomassandandits
itsimpact
impacton
onthe
thesolution
solutionsurface
surfacetension
tensionand
andpHpH
during the 4 days growth of A. platensis. The algal biomass increased with time, which increased
during the 4 days growth of A. platensis. The algal biomass increased with time, which increased thethe
pH and decreased the surface tension of the solution.
pH and decreased the surface tension of the solution.

Thedependence
The dependenceof offoaming
foamingcharacteristics,
characteristics, including
including FS,FS, FC,
FC, FE,
FE,andandFMD,
FMD,on onthethe
microalgal growth rate was monitored (Figure 6). FC increased steadily
microalgal growth rate was monitored (Figure 6). FC increased steadily with increasing with increasing
drybiomass
dry biomassweight
weightand andreached
reached1.2 1.2when
when1.51.5g/L
g/L dry
dry biomass
biomass was was produced
produced afterafter 44 days.
days.
AAsimilar
similartrend
trendwas
wasfound
foundforforFE
FEthat
thatincreased
increasedfrom
from1515toto4343when
whendry drybiomass
biomassofof1.51.5g/L
g/L
wasproduced
was producedafter after 4 days.
4 days. TheThe
foam foam produced
produced by 1.5by 1.5microalgal
g/L g/L microalgal dry biomass
dry biomass remained re-
mained
stable forstable for 10 s. Similarly,
10 s. Similarly, the FMD was the FMD was from
increased increased
0.02 to from
0.070.02
duringto 0.07 during the
the formation
formation
of of 1.5 g/L microalgal
1.5 g/L microalgal dry biomass. dry biomass.
The resultsTheshowed
results that
showed that all foaming
all foaming charac-
characteristics,
teristics, including FS, FC, FE, and FMD, were increased with the
including FS, FC, FE, and FMD, were increased with the increasing microalgal biomass increasing microalgal
biomass
dry weight.dry Bubble
weight.interfaces
Bubble interfaces were accumulated
were accumulated by microalgae
by microalgae together together withand
with EPS EPS
and formed
formed a soft aadhesive
soft adhesive gelfavoured
gel that that favoured the formation
the formation of stable ofand
stable and long-lasting
long-lasting foams,
foams,
as shown asin shown
Figure in4.Figure
As the4.microalgal
As the microalgal
density density increased,
increased, the cellsthewerecells were adsorbed
adsorbed by the
by the and
bubble bubble and carried
carried over to over to the surface
the surface of the solution
of the solution during aeration
during aeration or paddle or paddle
wheel
wheel rotation.
rotation. The EPSThe EPS excretion
excretion gradually gradually
decreased decreased thetension
the surface surfaceoftension of the because
the solution solution
eering 2022, 9, x FOR PEER REVIEW 7 of 12
Bioengineering 2022, 9, 257 7 of 11

because EPS behaved as a surfactant and increased the stickiness of the culture, therefore
EPS environment
creating a positive behaved as a surfactant
to generateand increased
stable the[33,37].
foaming stickiness of the culture, therefore creating a
positive environment to generate stable foaming [33,37].

Figure 6. The dependence

Figure 6. The of dependence
foaming characteristics, including foam
of foaming characteristics, capacity,
including foam
foam expansion,
capacity, foam expansion, foam
foam stability, and
stability, and foam maximum density, on the microalgal biomass dry weight for
foam maximum density, on the microalgal biomass dry weight produced 4
produced for 4 days.
days. Note: Foam Stability = Half-life of foam from generation to disappear, Foam capacity = Ratio
Note: Foam Stability = Half-life of foam from generation to disappear, Foam capacity = Ratio of
of foam volume to gas volume, Foam expansion = Ratio of foam volume to reduced liquid volume
foam volume to gas volume, Foam expansion = Ratio of foam volume to reduced liquid volume
from initial to final condition, and Foam maximum density = Ratio of the difference between the
fromand
initial liquid volume initial
theto final
final condition,
liquid volumeandwithFoam maximum
the final = Ratio
density Note:
foam volume. of the
Each datadifference
point between the
initialreplicates
is an average of three liquid volume
with aand the final
standard liquid volume
deviation, n = 3. with the final foam volume. Note: Each data point
is an average of three replicates with a standard deviation, n = 3.
3.3. Effect of CO2 Aeration on Foam Stability
3.3. Effect of CO2 Aeration on Foam Stability
Since CO2 gas influenced the rapid volume expansion of foam when the bioreactor
Since CO2 gas influenced the rapid volume expansion of foam when the bioreactor
was continuously wasaerated from the
continuously bottom,
aerated fromthethe
EPS attached
bottom, theto
EPStheattached
A. platensis
to thecellA.
wall
platensis cell wall
enhanced the formation of stable foams. The maximum solubility of CO 2 in an algal solu-
enhanced the formation of stable foams. The maximum solubility of CO2 in an algal
tion was essential for efficient
solution algal for
was essential growth,
efficientbutalgal
when the aeration
growth, but when ratethebecame
aeration greater
rate became greater
than the solubility capability of the solution, the supplied gas went through
than the solubility capability of the solution, the supplied gas went through the algal so- the algal
lution idly andsolution
contributedidly to thecontributed
and foam formation to theonfoamtheformation
surface of on thethe
solution.
surface of the solution.
The effect of CO The2 flow rate on the formation of foam was investigated by using the
effect of CO2 flow rate on the formation of foam was investigated by using the
Foamscan instrument
Foamscan that helped tothat
instrument analyze
helpedfour different
to analyze fourcharacteristics of the algal
different characteristics of the algal foam,
foam, including including the FS, FC, FE, and FMD. Figure 7a shows that the FS ofsolution
the FS, FC, FE, and FMD. Figure 7a shows that the FS of the the solution increased
increased gradually fromfrom
gradually 1 to 10 s during
1 to the 4 the
10 s during days4 as theas
days algal
the growth increased.
algal growth It alsoIt also showed
increased.
showed that FC andFC
that FEand
increased from 0.26
FE increased from ± 0.03
0.26 to
± 0.90
0.03 ±to0.03
0.90and 22 ±and
± 0.03 0.4622 to±380.46
± 0.46
to 38 ± 0.46 over
over time (Figuretime7a,b). These
(Figure results
7a,b). indicate
These results that CO2 aeration
indicate that COand 2
A. platensis
aeration and cell
A. den-
platensis cell density
sity increased simultaneously. FMD was the
increased simultaneously. FMD onlywasparameter
the only that decreased
parameter thatfrom 5.5 ± 2.8
decreased from 5.5 ± 2.8 to
to 3.1 × 10−2 ± 2.8
3.1 × 10−the
with 2 ± increasing
2.8 with theaeration
increasing rate of gasrate
aeration (Figure
of gas7b). This 7b).
(Figure could becould
This at- be attributed
tributed to the to
continuous supply of CO gas that led to rapid bubble formation
the continuous supply of CO2 gas that led to rapid bubble formation that ruptured more
2 that rup-
tured more quickly duedue
quickly to the high
to the aeration
high aeration rate. Thus,
rate. Thus, larger bubbles
larger bubbles with
withlower
lower density
density were formed
were formed atathigher
higherflowflowrates.
rates.
Bioengineering 2022,9,9,257
Bioengineering2022, x FOR PEER REVIEW 88 of
of 11
12

Figure7.7.Effects
Figure Effectsof
ofCO
CO22flow
flowrates
rateson
ondynamic
dynamiccharacteristics:
characteristics:(a)
(a)foam
foamstability
stabilityand
andexpansion
expansionand
and
(b) foam maximum density and capacity.
(b) foam maximum density and capacity.

3.4.
3.4.Effect
EffectofofCultivation
CultivationTemperature
Temperatureon
onFoaming
FoamingStability
Stability
Temperature
Temperatureis is oneone
of the
of most
the mostimportant control
important parameters
control during the
parameters algalthe
during growth,
algal
as the metabolic
growth, and enzymatic
as the metabolic activities are
and enzymatic directlyare
activities influenced by it duringby
directly influenced theit simultane-
during the
ous productionproduction
simultaneous of biomass and EPS [34,36,38].
of biomass and EPS A [34,36,38].
higher temperature
A higherreduces the solubility
temperature reduces
of gaseous components (i.e.,
the solubility of gaseous components CO 2 ) in the culture medium, whereas a lower temperature
(i.e., CO2) in the culture medium, whereas a lower
leads to reduced
temperature algal
leads growth and
to reduced algaldecreases
growth and the kinetics
decreasesof the
metabolic
kineticsactivities [38]. activi-
of metabolic
The
ties [38]. experimental data show that temperature greatly contributed to affecting the
foamingTheevents. A higher
experimental datatemperature affected the greatly
show that temperature bubble contributed
drag, bubble to rise velocity,
affecting the
surface
foamingtension,
events. and flow temperature
A higher behavior of the foam,the
affected which
bubble leddrag,
to anbubble
increased mixing and
rise velocity, sur-
gaseous holdand
face tension, up flow
within the bubbles.
behavior Thesewhich
of the foam, results
ledare in increased
to an accordance with and
mixing a previous
gaseous
study [18]. The foam film permeability increased with the increasing
hold up within the bubbles. These results are in accordance with a previous study temperature, whereas
[18].
the
Thesurface
foam filmtension decreased,
permeability as reported
increased withpreviously [39].temperature,
the increasing Temperaturewhereas
behavesthe assur-
an
indirect contributor to foaming events in photo-bioreactors. In Figure
face tension decreased, as reported previously [39]. Temperature behaves as an indirect 8a,b, it is shown
that foaming characteristics increased during the culture, such as FS from 0 to 9 s, FC from
Bioengineering 2022, 9, x FOR PEER REVIEW 9 of 12

Bioengineering 2022, 9, 257 9 of 11

contributor to foaming events in photo-bioreactors. In Figure 8a,b, it is shown that foam-

ing characteristics increased during the culture, such as FS from 0 to 9 s, FC from 0.28 ±
0.03±to0.03
0.28 0.03,±and
0.89to±0.89 0.03,FE
and FE from
from 26 ±to
26 ±−0.41 0.41
38to± 38 ± 0.41.
0.41. On theOnother
the other hand,
hand, onlyonly
2 ± 0.28 (Figure 8b). The decreased FMD could be
FMD FMD
de-
decreased from 6.1 ± 0.28 to 3.0 × 10
creased from 6.1 ± 0.28 to 3.0 × 10 ± 0.28 (Figure 8b). The decreased FMD could be due to
−2
due
the to
factthe facta that
that a higher
higher temperature
temperature resulted
resulted in theinformation
the formation of larger
of larger but dense
but less less dense
bub-
bubbles, which caused rapid drainage of liquid through the film-forming
bles, which caused rapid drainage of liquid through the film-forming unstable foam. unstable foam.

Figure8.8.Effects
Figure Effectsof
oftemperatures
temperatureson
ondynamic
dynamiccharacteristics:
characteristics:(a)
(a)foam
foamstability
stabilityand
andexpansion
expansionand
and
(b) foam maximum density and capacity.
(b) foam maximum density and capacity.

4.4.Conclusions
Conclusions
Thefoaming
The foamingmechanism
mechanism in in algal
algal culture
culture waswas investigated
investigated with with varying
varying CO2 flowCO2rates
flow
ratestemperature
and and temperature
usingusing a Foamscan
a Foamscan instrument.
instrument. It is It is concluded
concluded that,that, as the
as the microal-
microalgae
gae solution
solution became
became denser
denser and and reached
reached 2.4 g/L,
2.4 g/L, thethe adhesive
adhesive EPS
EPS released
released from
from A. A. platen-
platensis
sis decreased
decreased the the surface
surface tension
tension to 45
to 45 mN/m.Consequently,
mN/m. Consequently,ananincreased
increasedCO CO flowrate
2 2flow rate
of400
of 400mL/min
mL/min and a temperature of 40 40 ◦°C increased the
C increased the stability
stability of
of the
the foam
foam up up to
to 10
10ss
with 30 mm height and 1.8 mm bubble diameter. Based on the findings of this study, it is
Bioengineering 2022, 9, 257 10 of 11

recommended that a high temperature and rigorous CO2 aeration should be avoided to
mitigate the foaming problem in photobioreactors.

Author Contributions: Conceptualization, A.A.K., A.A., M.W.U. and J.C; methodology, A.A.K. and
A.A; software, A.A.K. and S.K.; validation, A.A.K. and K.F.S.A.; formal analysis, A.A.K. and S.K.;
investigation, A.A.K. and A.A.; resources, M.W.U. and J.C.; data curation, A.A.K., A.A. and K.F.S.A.;
writing—original draft preparation, A.A.K., A.A. and M.W.U.; writing—review and editing, M.W.U.,
S.H., M.I. and J.C.; visualization, A.A.K.; supervision, M.W.U. and J.C.; project administration, M.W.U.
and J.C.; funding acquisition, J.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was financially supported by the National Key Research and Development
Program of China (2017YFE0122800), Zhejiang Provincial Key Research and Development Program
of China (2020C04006), National Natural Science Foundation of China (21978120), Key R&D projects
in Jiangsu Province (BE2020405), the Natural Science Foundation of Jiangsu Province (BK20200889),
the Chinese Postdoctoral Science Foundation (2019M661761), and the Natural Science Foundation of
Jiangsu Higher Education Institutions (20KJB550003).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The researchers are thankful to the analysis and testing centers of Jiangsu
University, Zhenjiang, China, and Zhejiang University, Hangzhou, China, for the characterization of
different samples.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

References
1. Zhao, B.; Su, Y.; Zhang, Y.; Cui, G. Carbon Dioxide Fi Xation and Biomass Production from Combustion Fl Ue Gas Using Energy
Microalgae. Energy 2015, 89, 347–357. [CrossRef]
2. Ganesh Saratale, R.; Kumar, G.; Banu, R.; Xia, A.; Periyasamy, S.; Dattatraya Saratale, G. A Critical Review on Anaerobic Digestion
of Microalgae and Macroalgae and Co-Digestion of Biomass for Enhanced Methane Generation. Bioresour. Technol. 2018, 262,
319–332. [CrossRef] [PubMed]
3. Reen, S.; Chyuan, H.; Wayne, K.; Loke, P.; Phang, S.; Chuan, T.; Nagarajan, D.; Lee, D. Sustainable Approaches for Algae
Utilisation in Bioenergy Production. Renew. Energy 2017, 129, 838–852. [CrossRef]
4. Ho, S.; Chen, C.; Lee, D.; Chang, J. Perspectives on Microalgal CO 2 -Emission Mitigation Systems—A Review. Biotechnol. Adv.
2011, 29, 189–198. [CrossRef] [PubMed]
5. Razzak, S.A.; Hossain, M.M.; Lucky, R.A.; Bassi, A.S.; Lasa, H. De Integrated CO 2 Capture, Wastewater Treatment and Biofuel
Production by Microalgae Culturing—A Review. Renew. Sustain. Energy Rev. 2013, 27, 622–653. [CrossRef]
6. Khan, S.; Siddique, R.; Huanfei, D.; Shereen, M.A.; Nabi, G.; Bai, Q.; Manan, S.; Xue, M.; Ullah, M.W.; Bowen, H. Perspective
Applications and Associated Challenges of Using Nanocellulose in Treating Bone-Related Diseases. Front. Bioeng. Biotechnol.
2021, 9, 350. [CrossRef] [PubMed]
7. Ullah, M.W.; Manan, S.; Kiprono, S.J.; Ul-Islam, M.; Yang, G. Synthesis, Structure, and Properties of Bacterial Cellulose. In
Nanocellulose: From Fundamental to Advanced Materials; Huang, J., Lin, N., Dufresne, A., Eds.; Wiley: Weinheim, Germany, 2019; pp.
81–113. [CrossRef]
8. Pires, J.C.M.; Alvim-ferraz, M.C.M.; Martins, F.G. Photobioreactor Design for Microalgae Production through Computational Fl
Uid Dynamics: A Review. Renew. Sustain. Energy Rev. 2017, 79, 248–254. [CrossRef]
9. Lee, E.; Pruvost, J.; He, X.; Munipalli, R.; Pilon, L. Design Tool and Guidelines for Outdoor Photobioreactors. Chem. Eng. Sci. 2014,
106, 18–29. [CrossRef]
10. Spolaore, P.; Joannis-Cassan, C.; Duran, E.; Isambert, A. Commercial Applications of Microalgae. J. Biosci. Bioeng. 2006, 101, 87–96.
[CrossRef] [PubMed]
11. Prussi, M.; Buffi, M.; Casini, D.; Chiaramonti, D.; Martelli, F.; Carnevale, M.; Tredici, M.R. ScienceDirect Experimental and
Numerical Investigations of Mixing in Raceway Ponds for Algae Cultivation. Biomass Bioenergy 2014, 67, 390–400. [CrossRef]
12. Pankratz, S.; Oyedun, A.O.; Zhang, X.; Kumar, A. Algae Production Platforms for Canada ’ s Northern Climate. Renew. Sustain.
Energy Rev. 2017, 80, 109–120. [CrossRef]
Bioengineering 2022, 9, 257 11 of 11

13. de Godos, I.; Mendoza, J.L.; Acién, F.G.; Molina, E.; Banks, C.J.; Heaven, S.; Rogalla, F. Evaluation of Carbon Dioxide Mass Transfer
in Raceway Reactors for Microalgae Culture Using Flue Gases. Bioresour. Technol. 2014, 153, 307–314. [CrossRef] [PubMed]
14. Wang, J.; Nguyen, A.V.; Farrokhpay, S. A Critical Review of the Growth, Drainage and Collapse of Foams. Adv. Colloid Interface
Sci. 2016, 228, 55–70. [CrossRef] [PubMed]
15. Chen, X.W.; Yang, D.X.; Zou, Y.; Yang, X.Q. Stabilization and Functionalization of Aqueous Foams by Quillaja Saponin-Coated
Nanodroplets. Food Res. Int. 2017, 99, 679–687. [CrossRef] [PubMed]
16. Xu, L.; Xu, G.; Gong, H.; Dong, M.; Li, Y.; Zhou, Y. Colloids and Surfaces A: Physicochemical and Engineering Aspects Foam
Properties and Stabilizing Mechanism of Sodium Fatty Alcohol Polyoxyethylene Ether Sulfate-Welan Gum Composite Systems.
Colloids Surfaces A Physicochem. Eng. Asp. 2014, 456, 176–183. [CrossRef]
17. Kougias, P.G.; Boe, K.; Tsapekos, P.; Angelidaki, I. Bioresource Technology Foam Suppression in Overloaded Manure-Based
Biogas Reactors Using Antifoaming Agents. Bioresour. Technol. 2014, 153, 198–205. [CrossRef]
18. Subramanian, B.; Pagilla, K.R. Colloids and Surfaces B: Biointerfaces Mechanisms of Foam Formation in Anaerobic Digesters.
Colloids Surfaces B Biointerfaces 2015, 126, 621–630. [CrossRef]
19. Kougias, P.G.; Boe, K.; Einarsdottir, E.S.; Angelidaki, I. ScienceDirect Counteracting Foaming Caused by Lipids or Proteins in
Biogas Reactors Using Rapeseed Oil or Oleic Acid as Antifoaming Agents. Water Res. 2015, 79, 119–127. [CrossRef]
20. Moeller, L.; Lehnig, M.; Schenk, J.; Zehnsdorf, A. Bioresource Technology Foam Formation in Biogas Plants Caused by Anaerobic
Digestion of Sugar Beet. Bioresour. Technol. 2015, 178, 270–277. [CrossRef]
21. Kubar, A.A.; Cheng, J.; Kumar, S.; Liu, S.; Chen, S.; Tian, J. Strengthening Mass Transfer with the Tesla-Valve Baffles to Increase
the Biomass Yield of Arthrospira Platensis in a Column Photobioreactor. Bioresour. Technol. 2021, 320, 124337. [CrossRef]
22. Cheng, J.; Guo, W.; Song, Y.; Kumar, S.; Ameer Ali, K.; Zhou, J. Enhancing Vorticity Magnitude of Turbulent Flow to Promote
Photochemical Efficiency and Trichome Helix Pitch of Arthrospira Platensis in a Raceway Pond with Conic Baffles. Bioresour.
Technol. 2018, 269, 1–8. [CrossRef] [PubMed]
23. Ali Kubar, A.; Cheng, J.; Guo, W.; Kumar, S.; Song, Y. Development of a Single Helical Baffle to Increase CO2 Gas and Microalgal
Solution Mixing and Chlorella PY-ZU1 Biomass Yield. Bioresour. Technol. 2020, 307, 123253. [CrossRef] [PubMed]
24. Kumar, S.; Cheng, J.; Guo, W.; Ali, K.A.; Song, Y. Self-Rotary Propellers with Clockwise/Counterclockwise Blades Create Spiral
Flow Fields to Improve Mass Transfer and Promote Microalgae Growth. Bioresour. Technol. 2019, 286, 121384. [CrossRef] [PubMed]
25. Jones, S.A.; Laskaris, G.; Vincent-Bonnieu, S.; Farajzadeh, R.; Rossen, W.R. Effect of Surfactant Concentration on Foam: From
Coreflood Experiments to Implicit-Texture Foam-Model Parameters. J. Ind. Eng. Chem. 2016, 37, 268–276. [CrossRef]
26. Wang, H.; Guo, W.; Zheng, C.; Wang, D.; Zhan, H. Effect of Temperature on Foaming Ability and Foam Stability of Typical
Surfactants Used for Foaming Agent. J. Surfactants Deterg. 2017, 20, 615–622. [CrossRef]
27. Trabelsi, L.; M’sakni, N.H.; Ouada, H.B.; Bacha, H.; Roudesli, S. Partial Characterization of Extracellular Polysaccharides Produced
by Cyanobacterium Arthrospira Platensis. Biotechnol. Bioprocess Eng. 2009, 14, 27–31. [CrossRef]
28. Chentir, I.; Hamdi, M.; Doumandji, A.; HadjSadok, A.; Ouada, H.B.; Nasri, M.; Jridi, M. Enhancement of Extracellular Polymeric
Substances (EPS) Production in Spirulina (Arthrospira Sp.) by Two-Step Cultivation Process and Partial Characterization of Their
Polysaccharidic Moiety. Int. J. Biol. Macromol. 2017, 105, 1412–1420. [CrossRef]
29. Liu, Q.; Zhang, S.; Sun, D.; Xu, J. Foams Stabilized by Laponite Nanoparticles and Alkylammonium Bromides with Different
Alkyl Chain Lengths. Colloids Surfaces A Physicochem. Eng. Asp. 2010, 355, 151–157. [CrossRef]
30. Georgieva, D.; Cagna, A.; Langevin, D. Link between Surface Elasticity and Foam Stability. Soft Matter 2009, 5, 2063–2071.
[CrossRef]
31. Saint-Jalmes, A. Physical Chemistry in Foam Drainage and Coarsening. Soft Matter 2006, 2, 836. [CrossRef]
32. Roth, A.E.; Jones, C.D.; Durian, D.J. Bubble Statistics and Coarsening Dynamics for Quasi-Two-Dimensional Foams with
Increasing Liquid Content. Phys. Rev. E-Stat. Nonlinear Soft Matter Phys. 2013, 87, 1–14. [CrossRef] [PubMed]
33. Bernaerts, T.M.M.; Gheysen, L.; Foubert, I.; Hendrickx, M.E.; Van Loey, A.M. The Potential of Microalgae and Their Biopolymers
as Structuring Ingredients in Food: A Review. Biotechnol. Adv. 2019, 37, 107419. [CrossRef] [PubMed]
34. Singh, S.P.; Singh, P. Effect of Temperature and Light on the Growth of Algae Species: A Review. Renew. Sustain. Energy Rev. 2015,
50, 431–444. [CrossRef]
35. Ahmed, M.; Moerdijk-Poortvliet, T.C.W.; Wijnholds, A.; Stal, L.J.; Hasnain, S. Isolation, Characterization and Localization of
Extracellular Polymeric Substances from the Cyanobacterium Arthrospira Platensis Strain MMG-9. Eur. J. Phycol. 2014, 49,
143–150. [CrossRef]
36. Renaud, S.M.; Thinh, L.-V.; Lambrinidis, G.; Parry, D.L. Effect of Temperature on Growth, Chemical Composition and Fatty Acid
Composition of Tropical Australian Microalgae Grown in Batch Cultures. Aquaculture 2002, 211, 195–214. [CrossRef]
37. Mancuso Nichols, C.A.; Nairn, K.M.; Glattauer, V.; Blackburn, S.I.; Ramshaw, J.A.M.; Graham, L.D. Screening Microalgal Cultures
in Search of Microbial Exopolysaccharides with Potential as Adhesives. J. Adhes. 2009, 85, 97–125. [CrossRef]
38. Muñoz, R.; Guieysse, B. Algal-Bacterial Processes for the Treatment of Hazardous Contaminants: A Review. Water Res. 2006, 40,
2799–2815. [CrossRef]
39. Kapetas, L.; Vincent Bonnieu, S.; Danelis, S.; Rossen, W.R.; Farajzadeh, R.; Eftekhari, A.A.; Mohd Shafian, S.R.; Kamarul Bahrim,
R.Z. Effect of Temperature on Foam Flow in Porous Media. J. Ind. Eng. Chem. 2016, 36, 229–237. [CrossRef]