Biochemical Engineering Journal 151 (2019) 107312

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Computational fluid dynamic analysis of hydrodynamic shear stress T

generated by different impeller combinations in stirred bioreactor
Ritu Verma, Luv Mehan, Rahul Kumar, Arinjay Kumar, Aradhana Srivastava
⁎

University School of Chemical Technology, Guru Gobind Singh Indraprastha University, Sec-16C, Dwarka, New Delhi, 110078, India

HIGHLIGHTS

• Novel combination of impellers in bioreactor for microalgae growth.

• CFD analysis for hydrodynamic shear stress.
• Shear stress varies as 0.99–6.17 Pa with RPM change of 50–225.
• Shear sensitive microalgae cultivation.
• platensis shear stress optima as 2.64Pa at 100RPM producing 3.9g/l cells.

ARTICLE INFO ABSTRACT

Keywords: The production of microalgae has gained attention in food, health and biofuel industries. Optimized growth
Shear sensitive conditions such as agitation, aeration and nutrient concentration govern the hydrodynamic environment and
Microalgae sustainability of photoautotroph microalgae in a bioreactor. A numerical simulation using the k-epsilon model in
Impeller computational fluid dynamics (CFD) was used to investigate the hydrodynamic shear stress generated due to two
Hydrodynamic
combinations of impellers Rushton-Rushton and Rushton-marine in stirred bioreactor at different RPMs.
Bioreactor characteristics were modeled and simulated with different operational impeller speeds to compute the
shear stress generated in the fluid. The four-way flow regime in the fluid was generated by the angular motion of
Rushton-marine impellers. The rotation speeds ranging from 75 to 150 RPM exhibited shear stress of
1.97–3.96 Pa which is tolerable for large microalgae types. Further increase in RPM resulted in formation of
shear zones which was also verified experimentally by observing the growth of A. platensis at different rotational
speeds. The maximum biomass was achieved as 3.9 g/l at 100 RPM generating a shear stress of 2.64 Pa. The
observed growth of microalgae (34.46% higher) was attributed to the improved hydrodynamics in terms of shear
stress and mixing time in the bioreactor through altered impellers combination using the CFD approach.

1. Introduction effective internal mechanical agitation are the conventionally used

Microalgae find their use in production of single cell proteins, CO2
sequestration, wastewater treatment and biofuel production [1–4].
However, only a few out of the 30,000 known strains find their use in
industrial applications. It is essential to produce large microalgae bio-
mass for maximizing the products. The cultivation conditions such as
adequate nutrient concentration, pH and temperature and operating
conditions such as illumination intensities, agitation and aeration di-
rectly influence the growth of microalgae cells in the bioreactors [5].
The selection of cultivation system is based on effectiveness of the
above-mentioned parameters. The prime example of a reactor with
stirred-tank bioreactors. They are highly flexible to modifications and
provides high kLa (volumetric mass transfer coefficient) values for gas-
liquid transfer when pressurized air is supplied through the sparger.
The size of the gas bubbles and their dispersion throughout the tank are
critical to the performance of the bioreactor [6,7]. The dispersion of gas
in the medium is mainly the function of the impeller instead of the
sparger. The selection of impeller and its operation plays a major role in
the generation of flow regime in the reactor fluid which directly in-
fluences the growth of microalgae. The impeller must provide sufficient
rapid agitation to disperse the bubbles throughout the tank. It should
also provide increased residence time of the bubbles and nutrients

⁎
Corresponding author.
E-mail addresses: ritu15verma2@gmail.com (R. Verma), luvmehan@gmail.com (L. Mehan), rajput060895@gmail.com (R. Kumar),
arinjaykumar@gmail.com (A. Kumar), aradhana.usct@ipu.ac.in (A. Srivastava).

https://doi.org/10.1016/j.bej.2019.107312
Received 12 February 2019; Received in revised form 20 July 2019; Accepted 22 July 2019
Available online 23 July 2019
1369-703X/ © 2019 Elsevier B.V. All rights reserved.
R. Verma, et al.
within the liquid along with breaking larger bubbles into smaller ones.
However, inadequate stirring and multiple impeller systems are often
detrimental owing to the shear sensitivity exhibits by the microalgae
cells to varying degrees [8–10]. Although high hydrodynamic stress and
turbulence generated by the Rushton type impellers and larger bubbles
released from the ring type sparger in conventional bioreactors is fa-
vorable for the growth of various micro-organisms such as Tetraselmis
suecica (which can tolerate shear stress up to 80 Pa) but not suitable for
the shear sensitive microalgae species like Chaetoceros muelleri, Skele-
tonema costatum and Isochrysis galbana whose viability has been re-
ported to decrease gradually from 1.2 to 5.4 Pa [11]. Viability of
Chaetoceros muelleri is found to decrease by 52–66% at the shear stress
1–1.3 Pa [12]. However, shear rate of 7000 s−1 exhibits the highest
growth rate of Phaeodactylum tricornutum by maintaining a balance
between the gas-liquid mass transfer limitations and the shear stress
damage [13]. Shear sensitivity is dependent on hydrodynamics inside
the reactor and microalgae genotype. This is shown clearly in published
literature where damage to the Protoceratium reticulatum cells is ob-
served even at low shear rate of 0.12 s−1 [14]. The same is observed in
the case of Phaeodactylum tricornutum and Porphyridium cruentum where
the threshold impeller tip speed is beyond 1.56 m/s and between 2.45
and 2.89 m/s, respectively [15]. A co-axial cylindrical device when
used for the cultivation of Spirulina platensis under appropriate shear
stress environment causes cell damage at shear stress beyond 0.3 Pa
[16]. The reported data clearly indicates the importance of design
parameters in the cultivation of shear sensitive microalgae. A previous
work on a modified conventional bioreactor also describes the same
[17]. Further upgradation of the system is possible by involving im-
peller speed (RPM) and its effect on hydrodynamic shear stress and
microalgae growth inside the reactor. This analysis is essential to pre-
dict the optima for high cell density culture of shear sensitive micro-
algae cells. Therefore, present work was focused on the CFD modeling
and simulation of a stirred tank bioreactor (Bioflo™ 110) to analyze the
hydrodynamic shear stress generated at different rotational speeds
using different impeller combinations i.e. Rushton-Rushton, marine-
marine and Rushton-marine. The effects of different rotational speeds
and corresponding shear stress on the growth of A. platensis were also
experimentally studied. The simulation and experimental studies com-
bined provided the desirable rotational speed of combination of im-
pellers to be operated in the bioreactor for the generation of tolerable
shear stress to ensure efficient growth of microalgae.
2. Material and methods
2.1. Configuration of the conventional bioreactor
The conventional bioreactor (Bioflo™ 110, M/S Eppendorf, USA)
was initially equipped with two Rushton impellers (top) along with one
ring sparger (bottom) housed inside a 1 cm thick vessel of height
23.5 cm and internal diameter of 13.32 cm. The two impellers are
placed on the shaft at 13.3 cm and 6.5 cm distance from the base. The
sparger was placed beneath the impeller at a distance of 3.25 cm from
the base of the reactor. After the calculation of optimum spacing be-
tween the impellers, marine impeller replaced the bottom Rushton
impeller and sintered stainless steel microporous sparger replaced the
ring sparger [17]. Baffles were placed at the centre of the working
volume with the dimension of 12 cm × 1 cm × 0.2 cm (Height x Width
x Thickness).
Fig. 1 shows the CAD of the modified bioreactor including the above
components. These three-dimensional drawings were constructed with
the help of Autodesk SimStudio Tools 2016. The software uses Study
Design Environment as the UI engine with Auto desk CFD as a solver
component. Autodesk CFD utilizes standard κ-ε model which is the most
widely used isotropic two-equation turbulence model for steady in-
compressible turbulent flow in other commercial software programs. It
is the most appropriate and robust model for the small stirred tank
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Biochemical Engineering Journal 151 (2019) 107312
bioreactors which have supreme well mixed zones in the vessel. It
provides equitable accuracy in results for an extensive range of turbu-
lent flows [18,19]. Its stable solution makes it preferable over other
highly refined models including shear stress transport (SST) which is
considered in case of low Re number. This software package is freely
available for students with an educational license (https://www.
autodesk.com/education/free-software/cfd). Fig. 1(a)–(c) depicts the
front, top and bottom view of the complete agitation and aeration as-
sembly. Rushton impeller has a diameter of 5.5 cm with blades di-
mensions as 1.5 cm x 1.8 cm. Marine impeller has a diameter of 7 cm
with 45° angular blades. Both the impellers are attached to the same
shaft which is connected to the top driven motor and enters from the
upper side of bioreactor. The cylindrical sintered stainless steel micro-
porous sparger has 5.8 × 108 pores of the size 0.2 μm each. The total
length of the cylindrical sparger is 3.78 cm on which the pores are
distributed along the length of 2.18 cm and placed centrally beneath the
lower impeller. Continuous sterile air was supplied to the reactor at a
superficial velocity of 0.0016 m/s. The working volume was fixed at
2.2 l (i.e. 2/3rd of the bioreactor volume) and filled with water as it has
negligible viscosity difference with broth at 28+1 °C.
CFD simulations were carried out and verified by experimental
work. Different arrangement and configuration of Rushton and Marine
impeller were simulated via CFD at different RPM i.e. 50, 75, 100, 125,
150, 175, 200 and 225 whereas 100 RPM was chosen as the control
RPM further studies to obtain the better arrangement and configuration
of Rushton and marine impeller.
2.2. Strain used and cultivation conditions
Arthrospira platensis supplied from Algal Research and Supply
(Carlsbad, USA) was used for the experimental studies carried out for
the verification of simulation results. A. platensis was selected as well-
suited shear sensitive specie due to its high protein content and other
industrial benefits. It has abridged trichrome chains that contribute to
its low shear tolerance but high mass transfer and illumination ex-
posure capabilities [16]. It exhibits steady growth rate even at the
sudden alteration in the shear stress due to regeneration of the tri-
chromes [20]. It reveals higher growth rate on gentle aeration (0.2–1.2
vvm) and doesn’t show a very significant effect on further increments in
aeration [21]. Increase in shear stress (up to 0.6 Pa) increases photo-
synthetic activity of cells even at low illumination intensity. Turbulent
motion makes the cells move about to their helix that permits illumi-
nation to scope pigments generation which generally shaded by helical
structure [16].
In present study, the culture was maintained and preserved using
the methodology described elsewhere [17]. The experimental studies
were performed by cultivating A. platensis at various rotational speeds
of RPM 50, 75, 100, 125, 150, 175, 200 and 225 using two combina-
tions of impellers to analyse the effects of respective shear stress on the
growth of microalgae. The entire methodology was same in both the
fermentation runs. A. platensis was first cultivated in the conventional
bioreactor BIOFLO 110 having two Rushton type impellers and then in
one with the combination of Rushton-marine impellers. Cultivations
were carried out to compare the performance of different impeller
combinations in terms of the biomass build up. Bioreactor with mod-
ified Zarrouk’s medium [17] was autoclaved and inoculated with 10%
(v/v) inoculum. The illumination intensity of 27, 284 lx was measured
at the reactor surface using HTC instrument, LX-103 Light meter Lux-
meter. Temperature, pH and photoperiod were maintained as 30 °C, 9
and 12:12 (Light: Dark) throughout the batch fermentation, respec-
tively. Continuous sterile air was supplied at the rate of 1.36 LPM
(0.62vvm) through micro porous sparger. CO2 from air was used as
carbon substrate for microalgae growth under phototrophic conditions.
R. Verma, et al.
Fig. 1. (a) Front view of the fermenter incorporated with Rushton and marine impeller; and sintered micro-porous sparger (b) top view of the fermenter (c) bottom
view of the fermenter.
2.3. Assay methods
2.3.1. Growth analysis
Samples were withdrawn after every 24 h for growth analysis. The
growth was analysed as absorbance at 680 nm and the dry cell con-
centration was measured using gravimetric method. 5 ml sample was
poured into an already dried and weighed foil cup. It was then kept at
70 °C for 12 h. Dried cup with biomass was weighed to obtain dry
biomass concentration.
2.3.2. Nitrate estimation
The residual nitrate concentration was measured using Ultraviolet
Spectrophotometric method [22]. 2 ml of collected sample was cen-
trifuged for 20 min at 8000 RPM. Supernatant was diluted to 1000-folds
to bring the concentration in the range of standard curve. 1 ml of 1 N
HCl was added to 5 ml of diluted supernatant to restrict the interference
of hydroxides and carbonates. Absorbance was measured at 220 nm and
275 nm for the analysis of nitrate and organic content respectively. The
actual nitrate content was calculated using the Eq. 1. Standard curve
was plotted using the sodium nitrate as the nitrate content.
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Biochemical Engineering Journal 151 (2019) 107312
Residual nitrate=Absorbance@220nm – 2(Absorbance@275nm) (1)
All sets of experiments were performed in triplicates. And average
values of biomass concentration are given in figures.
3. Theory and calculation
3.1. CFD model governing equations
The specific material properties to specific parts of the bioreactor
such as: glass to the bioreactor vessel; steel to all interiors such as pi-
pelines, baffles, impellers, sparger, shaft; water to rest of the working
volume space were applied in this simulation. The pressure at the
surface of water in the reactor was kept at 1.02 atm. The temperature of
the vessel was kept constant at 28ºC. At this temperature, the surface
tension and viscosity of water air system was kept at 7.15 × 10 2 N/m
and 0.8324 mPa.s, respectively. All other physical properties of water
were selected automatically by the software for the given temperature.
Water played the part of the primary phase in this study whereas air
played its role as the secondary phase and no other phase was present in
R. Verma, et al. Biochemical Engineering Journal 151 (2019) 107312

Fig. 2. Single layered meshed diagram of the fermenter (Layer factor = 0.45 and gradation = 1.1).

the domain that could represent biological cells. The viscosity was ( U)
+ (µeff U ) = . + . (µeff U )T + B
measured for all the sampled broth using LVDV-II + PRO Viscometer t (7)
(Brookfield Engineering Laboratories, USA). The observed viscosity
exhibited the variation with water as 0.01 mPa.s. The velocity of the The Turbulent Kinetic Energy (TKE) equation (Eq. 8) and the
water content was kept at 0 cm/s initially as there were no inflows and Turbulent Energy Dissipation (TED) equation (Eq. 9) [24]:
outflows. The angular motion was specified at 50, 75, 100, 125, 150,
175, 200 and 225 RPM in both the impellers for distinct simulations. ( k) µt
+ . Uk = . µ+ k + Pk
The presence and action of distinct impellers in the domain generated t k (8)
different flow regimes. The governing equations of the model are given
as follows.
( ) µt
The Continuity Equation [23]: + . U = . µ+ + Pk + (C 1 Pk C2 )
t k (9)
+ . ( U) = 0
t (2) Where constants, C 1 = 1.44, C 2 = 1.92, σκ = 1 and σε = 1.3.

Where ρ is the density, t is the time and U is the fluid particle velocity.
For constant density fluid (incompressible fluid), ρ is independent of 3.2. Meshing
space and time.

.U=0 (3) Enhanced mesh with a single layer of layer factor of 0.45 and layer
gradation of 1.1 is given in Fig. 2. The meshing resulted in 25,984
The Navier-Stokes equation [23]: numbers of elements along with 5458 number of nodes. Three different
2U grid sizes of 10,000, 20,000 and 30,000 were selected initially to per-
DUj P
form simulations and compare the results. The purpose was to find if
j
= µ
Dt xi xi xj xj (4) negligible variation in result exists otherwise further increase the grid
size.
Under the assumption that μ and ρ are uniform and . U = 0 , where D
Dt
is the time derivative, μ is the coefficient of viscosity, P is the pressure
and ᴪ is the gravitational potential.
4. Results
The constant eddy viscosity model equations:

K2 Simulations carried out at three different grid sizes i.e. 10,000,

µt = Cµ 20,000 and 30,000, were considered as scenario-1, scenario-2 and
(5)
scenario-3 respectively. The variation in the results of scenario 1 and
Cp scenario 2 was found to be 5.2%, whereas further increase in the grid
Kt = µt size by 10k reflected variation as 0.93%. The steady state was obtained
t (6)
between the iterations 7000–8000 for all angular velocities for all
Where μt is dynamic eddy viscosity, is density, is turbulence dis- scenarios. All the simulations were stopped at 10,000 iterations. CPU
sipation rate, K is turbulent kinetic energy, t is a turbulent Prandtl for the simulation of three different combinations of Rushton-Rushton,
number (usually taken to be 1.0) and Cμ is an empirical constant. Rushton-marine and marine-marine at 100RPM was 94413, 92,839 and
The Momentum Equation: 91984 s respectively.

4
R. Verma, et al. Biochemical Engineering Journal 151 (2019) 107312

Fig. 3. Cross sectional view from top across height of (a) single Rushton impeller and (b) single marine impeller; and (c) Flow regime using impeller combinations
(c.1) Rushton-Rushton, (c.2) Rushton-marine and (c.3) marine-marine at 100RPM.

Table 1 4.1. Comparison between agitation systems in the bioreactor

Shear stress obtained from CFD simulations and biomass build-up (Provided
with S.D values) in A. platensis cultivation carried out at different RPMs using Cross sectional view of velocity profile and flow regime in the case
two agitation systems i.e. Rushton-Rushton and Rushton-marine impeller. of single Rushton impeller is shown in Fig. 3(a) where the blue color
Rotational Rushton-Rushton Rushton-marine depicts the non-shear zones, green and yellow color show the enhan-
speed, RPM cing shear zone areas and red colored zones are not suitable for shear
Simulated Biomass Simulated Biomass sensitive cells. The maximum shear for a rotational speed is generated
shear stress, Concentration, shear stress, Concentration,
near the blades of the impeller when in motion. Fig. 3(b) shows the flow
Pa g/l Pa g/l
regime generated due to single marine impeller in the reactor. In case of
50 1.80 1.1 ± 0.02 0.99 2.2 ± 0.06 marine impeller, the maximum shear for a rotational speed is generated
75 3.03 1.3 ± 0.05 1.97 2.9 ± 0.04 in the direction away from the tip of its blades.
100 3.82 2.4 ± 0.07 2.64 3.9 ± 0.03
Fig. 3(c) shows the comparison of three flow regimes generated
125 6.01 1.72 ± 0.03 3.32 3.87 ± 0.05
150 7.5 1.09 ± 0.01 3.96 3.81 ± 0.02 using the three combinations of impellers i.e. Rushton-Rushton,
175 10.17 0.93 ± 0.008 4.64 3.2 ± 0.07 Rushton-marine and marine-marine at 100 RPM. Stagnant zones of
200 13.74 0.89 ± 0.04 5.60 2.8 ± 0.01 varying sizes are visible near the walls and at the bottom for each im-
225 16.65 0.8 ± 0.06 6.17 2.6 ± 0.05 peller combination. The size of stagnant zones depends upon the im-
peller geometry which in turn directly impacts the position and

5
R. Verma, et al. Biochemical Engineering Journal 151 (2019) 107312

Fig. 4. Vertical cross-sectional view of the modified conventional bioreactor equipped with Rushton-marine impellers at (a) 225 RPM, (b) 200 RPM, (c) 175 RPM, (d)
150 RPM, (e) 125 RPM, (f) 100 RPM, (g) 75 RPM, (h) 50 RPM showing velocity magnitude of 0–66.66 cm/s and temperature varying from 27.72 to 28.28 °C.

magnitude of the shear stress. Among all impeller geometric para-
meters, the impeller diameter and the blade angle highly influence the
shear stress magnitude. The mixing time from the simulation results
were calculated at the time step number when the turbulent kinetic
energy and turbulent energy dissipation saturated and achieved steady
state in the convergence plot. The mixing time resulted from the si-
mulations for Rushton-Rushton, Rushton-marine and marine-marine
were 11.2, 9.9 and 5.2 s, respectively. Although marine-marine com-
bination exhibited acceptable shear stress value of 2.67 Pa and mixing
time but it provided lower volume coverage for mixing and the larger
stagnant zones as compared to other two combinations. Therefore, this
combination was not considered for further studies. Rushton-Rushton
and Rushton-marine generated the shear stress of 3.82 and 2.64 Pa,
respectively. Attributing to better mixing by producing least stagnant
zones and acceptable shear stress in the reactor, combination of
Rushton-Rushton and Rushton-marine were used for further studies.
The hydrodynamic parameters generated from these three simulations
(Fig. 3) exhibited the potential of Rushton-Rushton and Rushton-marine

6
R. Verma, et al.
Fig. 5. Simulation and experimental results obtained at different RPMs varying from 50 to 225 for (a) Rushton-Rushton, (b) Rushton-marine and (c) the overall
analysis at 100 RPM.
for better growth of microalgae.
4.2. CFD and experimental analysis of shear stress in the bioreactor
The combinations of Rushton-Rushton and Rushton-marine im-
pellers were further used for CFD simulations at all range of rotational
speeds and for experimental studies. The results given in Table 1 were
obtained on the completion of 10,000 iterations and provided a com-
parison of averaged hydrodynamic shear stress in the complete fluid
domain generated at different RPMs using both the impeller combina-
tions. The increase in rotational speed (RPM) resulted in the increase in
velocity magnitude and the energy incorporated.
Shear stress at different RPM generated in the reactor equipped with
Rushton-Rushton is approximately twice as that of Rushton-marine
(Table 1). The modified bioreactor assembly equipped with the com-
bination of Rushton-marine impellers allowed better extent to homo-
genous mixing by considering the mixing time, volume coverage for
mixing and stagnant zones. Fig. 4 shows the four-way flow regime
generated inside the bioreactor equipped with Rushton-marine im-
pellers at different RPM and temperature range. Comparing C.1 and C.2
of Fig. 3, the major volume coverage for mixing is observed with
Rushton-marine combination. The velocity in the range 6.66–19.99 cm/
s at the base of the reactor clearly depicts the effective functioning of
the marine impeller. This prevents the settling of the cells. Under the
assumption of no slip condition and no pressure gradient this simula-
tion study is an example of pure shear driven flow. The temperature of
the impeller shaft and the wall of the reactor were at 28 °C as a
boundary condition for the simulations. Viscous dissipation acts as a
source of thermal energy that causes temperature gradient as no heat
conduction is happening. The viscous dissipation is directly propor-
tional to the square of the velocity gradient. The temperature gradient
was extremely small in all batches of microalgae cultivation and so the
viscous dissipation was negligible in the current study. The viscous
dissipation won’t be important in scale-up of the same bioreactor as the
distance between the impeller blades and wall is large enough for this
lab scale reactor. However, viscous dissipation makes relevance if more
viscous fluids and a narrow space between the two boundaries exist. As
shown on the velocity magnitude scale, blue color depicts the shear less
zone and up the scale to green, yellow and red show elevating shear
zones. The profiles in Fig. 4 also reveal that most of the shear sensitive
cells are sustainable up to 150 RPM exhibiting colors blue to yellow and
corresponding shear stress up to 3.96 Pa. However, the shear sensitivity
depends on specific strain type; as few strains reported to sustain at
higher RPMs. The red color might be unsuitable for numerous shear
sensitive microalgae as it is visible at 200 RPM and above.
The experimental studies were also performed for verification of the
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Biochemical Engineering Journal 151 (2019) 107312
results from CFD simulations and to compare the two selected combi-
nations of impellers i.e. Rushton-Rushton and Rushton-marine. Eight
batch fermentations were carried out at different RPMs for each im-
peller combination. The effect of shear stress was studied in the terms of
growth maxima of A. platensis and the results are appended in Table 1
which clearly shows that combination of Rushton-marine impeller is
more efficient than two Rushton impellers in terms of biomass con-
centration at 100 RPM. Low biomass concentration of 2.2 g/l was
achieved in the case of Rushton-marine impeller combination at a 50
RPM because of settling of cells at the bottom of the reactor. This value
was further increased in batches carried out at 75 and 100 RPM. The
maximum biomass concentration of 3.9 g/l was achieved at 100 RPM
consuming 95% of the nitrates present in the modified Zarrouk’s
medium.
Fig. 5 is highlighting the overall result analysis of the two impeller
combinations i.e. Rushton-Rushton and Rushton-marine at 100RPM.
Two Rushton impellers required 26.6% higher gassed power than that
of other combination. The maximum biomass concentration achieved
with Rushton-Rushton was 38.46% lower than that achieved with
Rushton-marine combination. In brief, the comparison in Fig. 5 high-
lights that Rushton-marine combination exhibited lower mixing time,
power consumption and shear stress but higher biomass build up than
that of Rushton-Rushton.
5. Discussion
5.1. Comparison between the two combinations of impellers in the
bioreactor
Rushton impellers are known for providing a radial flow of the li-
quid and hence pushing the medium towards the reactor walls. As the
rotational speed increases, the spread towards the wall increases and
hence larger shear zones are formed. Whereas, marine impellers pro-
vide axial motion of the liquid in the reactor and circulate the liquid
towards axis and then raise it along the walls. The enhanced rotational
speed reduces settling of biomass and does not contribute in the for-
mation of intolerable shear zones. The periphery of fluid flow by axial
impellers forms bigger radius of mixing zones as compared to that of the
Rushton impeller [17]. Shear stress depends on the impeller geometric
parameters as well as on the impeller rotational speed [25]. The results
obtained from CFD simulations also exhibited that shear stress values
increase with increase in the impeller rotational speed and the number
of impeller blades. The combination of Rushton-Rushton impeller
having 6 blades configured at right angle generated high shear stress
values as compared to marine-marine combination having 3 blades
angled at 45°.
R. Verma, et al. Biochemical Engineering Journal 151 (2019) 107312

Table 2
Microalgae growth in stirred tank bioreactor with different impeller type and their hydrodynamics.
Ref. Reactor configuration Microalgae type and Hydrodynamic Growth results Inferences and remarks
their properties conditions

[30] Stirred tank

Agitator- Single
Chlorella vulgaris
Green algae
Tip speed-
0-5.89m/s
Biomass concentration-1.48g/l • Effect of hydrodynamic stress on the
photosynthetic activity of cells
spinning plate
Slightly shear tolerant Shear stress-
• Tip velocity and the shear stress on wall affects
cellular activities and may also become cause of
Unicellular 0-3.66 Pa death at high shear stress
Scenedesmus obliquus
Green algae
Tip speed-
0-5.89m/s
Biomass concentration-0.77g/l • Flow regime- Axial motion within the
periphery of spinning plate
Slightly shear tolerant
4-cell colonies
Shear stress-
0-3.66 Pa
• Order of shear tolerance- C. vulgaris > S.
obliquus > C. reinhardtii > A.flos-aquae
C. reinhardtii
Green algae
Tip speed-
0-5.89m/s
Biomass concentration-0.84 g/l
Speicfic growth rate- 0.012 h−1
• A.flos-aquae compeltely damaged at 0.45Pa
Shear sensitive Shear stress-
Unicellular 0-3.66 Pa
A.flos-aquae Tip speed- Biomass concentration- 0.22 g/l
Cyanobacteria 0-1.26m/s
Shear sensitive Shear stress-
Filamentous 0-0.45 Pa
[20] Stirred tank C. reinhardtii Tip speed-0.79-7.85
m/s
Speicfic growth rate- 0.11 h−1 • Tip
growth
speed > 6.28m/s causes decreased

Agitator- Multiple Green algae • Flow regime- Radial motion

vertical bladed turbine
stirrer
• Radial flow regime exibited better growth as
compared to axial in case of C. reinhardtii
Shear sensitive
Unicellular
• Filamentous specie is more sensitive as
compared to unicellular type
S. platensis Tip speed-0.79-
7.85m/s
Speicfic growth rate- 0.07 h−1 • S.lowplatensis being filamentous specie requires
tip speed
Cyanobacteria
Shear sensitive
Filamentous
[31] Stirred tank P. cruentem Tip speed- 0.68-
2.89m/s
Biomass concentration-1.03 g/l • Threshold
2.89m/s
tip speed is between 2.45 and

Agitator- 2 four bladed Red algae

turbines
Shear sensitive
Unicellular
[32] Stirred tank S. platensis Tip speed- 1.1m/s Biomass concentration- 4.44g/l • Flow regime- radial motion
Agitator- Gas inducing
4-bladed impeller
Cyanobacteria • Low biomass productivity at high tip speed
Shear sensitive
Filamentous
[14] Stirred tank P. reticulatum Tip speed- 0.288m/s Biomass concentration- 2.5×104
cells/ml
• Maximum permissible RPM suggested as 50
to avoid shear stress effects.
Agitator- 3 bladed
impeller
Dinoflagellate • P.requires
reticulatum being highly shear sensitive
low tip speed
Non baffled Shear sensitive
[33] *
Shaken flask P. reticulatum Shear stress- 0.00016
Pa
– • Cell shear sensitivity is higher in dark phase
Agitation- orbital shaker Dinoflagellate
Shear sensitive
[34] *
Couette flow and shaken
flask
I.polyedrum Shear stress-0.004 Pa Biomass concentration-3.25×103
cells/ml
• Turbulent
growth
conditions reduces dinoflgellate

Highly shear sensitive

Red tide dinoflagellate
• Response to turbulence depend on
environemntal and physiological factors
Present work Stirred tank A.platensis
Cyanobacteria
Tip speed- 0.33m/s
Shear stress- 2.64 Pa
Biomass concentration-3.9g/l • Combination of radial alongwith axial
motion exhibited high growth even at low tip
Shear sensitive speeds
Filamentous

* Note: Highly shear sensitive microalgae such as P. reticulatum, L. polyedrum are not capable to sustain stress generated in stirred tank configuration so grown in
shaken flask [33,34].

It was important to have an agitation assembly that provides ef-
fective mixing because the cultivation of microalgae requires the gas to
remain in the bioreactor periphery for longer duration. Higher mixing
time of 9.9 s for Rushton-marine and 11.2 s for Rushton-Rushton im-
pellers were obtained which resulted in the movement of gas in the
medium for longer duration. These results from CFD analysis motivated
the authors to study hydrodynamic shear stress effects on A. platensis
with the combination of Rushton-Rushton and Rushton-marine im-
pellers in the bioreactor. In case of two Rushton impellers, the cells
settled down at the bottom of the reactor as larger stagnant zones were
observed during fermentation. There was no peripheral movement of
cells in the reactor. Brown dead cells were also observed at the bottom
after 3–5 days which further increased with time.
5.2. CFD and experimental analysis of shear stress in the bioreactor
The major problems in the cultivation of shear sensitive microalgae
in bioreactor were settling of the cells to the bottom, stagnant zone
formation and cell damage. As Rushton impeller has been proven as the
main contributor to the high shear stress and settling of cells, one

8
R. Verma, et al.
Rushton impeller at bottom was replaced with the marine impeller. A
combination of Rushton-marine impeller provided lesser shear en-
vironment in the bioreactor and favorable hydrodynamic conditions
when compared to that with the combination of two Rushton impellers.
This modified conventional bioreactor would also be suitable for cul-
tivating C. muelleri, S. costatum and I. galbana at an RPM of 150 and 175
which are reported to withstand the shear stress of 1.2–5.4 Pa [11]. A
study on Cyanobacteria shows that a shear rate value of 2.2 s−1 can
decrease its ability of carbon fixation and nitrate metabolism [26]. On
the basis of published literatures on tolerable shear rate for various
microbial cells, the authors believe that the designed assembly and
optimized operational conditions would be appropriate for not only
microalgae cells but for other shear sensitive cells as well [27–29].
Batch fermentations were carried out to analyze the effects of shear
stress generated due to the combination of Rushton-Rushton and
Rushton-marine impellers at various speeds on the growth of A. pla-
tensis. The Rushton-Rushton impellers generated higher shear stress and
lower biomass concentration at all range of RPM than that generated by
the combination of Rushton-marine impeller (Table 1). Although, al-
most similar results of biomass were attained in the range of 100–150
RPM with Rushton-marine impeller combination but further increase in
impeller speed resulted in a decrease in biomass concentration due to
high hydrodynamic shear stress. In the case of Rushton-marine im-
pellers, dead cells were also observed in the samples collected after 3–4
days at 200 RPM and above due to vigorous mixing. These experimental
studies clearly supported the results obtained from CFD simulations.
The impeller speed range of 100–150 RPM causing shear stress as
2.64–3.96 Pa exhibited nearly comparable maximum biomass con-
centration but 100 RPM exhibiting the highest biomass among all re-
sulted as the most appropriate impeller speed for the growth of A.
platensis.
5.3. Validation of simulations
Simulation results in terms of hydrodynamic shear stress and tip
speed and experimental results in terms of biomass concentration were
qualitatively and quantitatively compared for stirred tank bioreactor
used in the present work with the previously published studies on si-
milar bioreactor (Table 2). Studies on stirred tank bioreactors with
impeller types such as spinning plate, gas induced 4-bladed impeller; 4-
bladed turbines and multiple vertical bladed turbine stirrers reported
hydrodynamic shear stress of the order of 0–3.66 Pa. Similarly, present
work on stirred tank bioreactor with combination of Rushton-marine
impellers produced hydrodynamic stress range up to 0.99–2.64 Pa for
optimum growth which validates the present study. The tip speed in the
published literature ranges from 0 to 7.85 m/s which is a quite high
value as compared to 0.33 m/s used in present work. The flow regime
generated due to combined radial and axial flow even at low tip speed
has efficiently improved the mixing and provided a highest biomass of
3.9 g/l in 5 days at 100RPM.
5.4. Conclusion
In conclusion, the impacts of combination of impellers on the hy-
drodynamic shear stress in the bioreactor were quantitatively assessed
using the CFD simulation and its validation was performed through the
experiments carried out at different RPMs. The combined radial and
axial motion produced unique flow regime for generating lesser shear
stress and complete mixing. The selected combination of impellers in
CFD analysis has successfully provided the clear view of stagnant zones
and flow regimes in the bioreactor fluid. The bioreactor was modified
and the optimum rotational speed of 100RPM was achieved for the
cultivation of shear sensitive microalgae. It was not possible to main-
tain good amount of mass transfer without any mechanical agitation.
The combination of Rushton-marine impeller in Bioflo™ 110 promises
homogeneity throughout the working volume of the bioreactor. This
9
Biochemical Engineering Journal 151 (2019) 107312
combination also resulted lesser power input as compared to that of
Rushton-Rushton placed in the conventional bioreactor. The angular
(radial and axial) motion of both the impellers fixed at 75–150 RPM
exhibited shear stress of 1.97–3.96 Pa which promotes numerous mi-
croalgae growth [30–32]. Operating the reactor at 100 RPM not only
make the hydrodynamic environment more suitable for A. platensis but
also keeps its production feasible even at 2.64 Pa by building up 3.9 g/l
of cells. Enhanced cell production was due to the combination of
homogeneity and appropriate shear stress generated during cultivation.
The simulation can be further extended for bioreactors with high aspect
ratio by providing multiple sets of Rushton-marine combination. The
present studies are also useful for growing all type of shear sensitive
unicellular or multicellular microbes.
Declaration of Competing Interest
None.
Acknowledgements
We are thankful to University School of Chemical Technology, Guru
Gobind Singh Indraprastha University, New Delhi, for all the lab fa-
cilities and infrastructure provided. We acknowledge the university
support for the same.
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