Biochemical Engineering Journal 191 (2023) 108794

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Biochemical Engineering Journal

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

Design and engineering characterization of a horizontal tubular bioreactor

with spiral impeller for cell cultivation
Rajesh Sharma , Wesley Collair , Aqeel Williams , Susan T.L. Harrison , Siew L. Tai *
Centre for Bioprocess Engineering Research, Department of Chemical Engineering, Faculty of Engineering and the Built Environment, University of Cape Town, Private
Bag, Rondebosch 7701, South Africa

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

Keywords: Aerated stirred tank bioreactors (STRs) are the norm for commercial production of biologicals but tend to exert
Bioreactor design high shear forces from high impeller tip speeds, direct sparging, bubble disruption at the surface and foam
Minimum agitation speed formation. In this work, a novel 5 liter horizontal tubular bioreactor (HTB) fitted with a spiral impeller was
Fluid flow pattern
designed for cell cultures. The abiotic and engineering characterization of the HTB was undertaken in terms of
Mixing time
Oxygen mass transfer
fluid flow pattern, mixing time (θm ), minimum agitation speed Njs, volumetric oxygen mass transfer coefficient
Power consumption (kL a), and power consumption (P). The bioreactor showed optimum operability between 2 and 3 litre, and
comparable mixing time ranges, power consumption and volumetric oxygen mass transfer coefficients (kL a)
between 5 and 16 h− 1 to a STR and other conventional systems. An ANOVA analysis disseminated that impeller
immersion and mixing speeds had significant effects on mixing times, while aeration rates and mixing speeds
significantly impacted kL a. The abiotic and engineering characterization investigations revealed that the
bioreactor design supported good surface aeration with bubble entrainment for oxygen mass transfer while
imparting low energy into the system, which are key attributes in promoting a low shear environment for cell
culturing.

improved control of the microenvironment to suppress programmed cell

death [4]. No bioreactor design has shown complete success for every
1. Introduction
cell line but stirred tank bioreactors have become the industry choice
because of its proven and well-established track record for upstream
The first commercial stirred tank bioreactor used for mammalian cell
process development and scale up [5]. An ongoing concern with STRs
culture technology was commissioned to produce polio vaccine in baby
for cell culture is the minimising of shear forces.
hamster kidney cells (BHK) in the 1960s [1,2]. The bioreactor designed
Cell cultures require oxygen for their growth and metabolism but
optimally for its task provides an optimum growth condition for cell
have lower oxygen demand than bacterial and yeast cultures. The oxy­
culture through efficient mixing, optimum mass and heat transfer,
gen mass transfer coefficient(kL a) of sparged stirred tank bioreactor
controlled growth environment such as pH, temperature and, particu­
under operating conditions used to grow mammalian cells typically falls
larly for mammalian cell culture, low shear conditions. The reactor
between 1 and 15 h− 1 [2]. High-density culture requires a larger oxygen
should be easy to scale-up and avoid spatial gradients caused by
supply, which is often not met by surface oxygenation methods alone
improper mixing which create a heterogeneous environment. Such a
[6], and often oxygen then becomes a limiting factor. Sparging of gases
heterogeneous environment can cause negative effects on cell viability,
directly into the bioreactor supplements oxygen requirement but this
metabolism, protein expression, and longevity of the culture [3].
method has been reported to damage the mammalian cells [2,7]. The
For cell culture, ongoing studies to improve bioreactor design have
oxygen uptake rate (OUR) of cell culture determines the desired oxygen
addressed lower energy utilization, increased mass transfer efficiency,
transfer rate (OTR) threshold of a system to support the growing cells,
improved mixing, real-time monitoring of cell growth and viability, and

Abbreviations: 3D, Three dimensions; BHK, Baby hamster Kidney; CFD, Computational fluid dynamics; CSV, Comma-separated values; DMA, Dynamic membrane
aeration; DO, Dissolved oxygen; HTB, Horizontal tubular bioreactor; LPM, Liter per minute (L min− 1); MFC, Mass flow controller; OD, Outer diameter; OSR, Orbitally
shaken reactor; OTR, Oxygen transfer rate (mmol O2 L− 1 h− 1); OUR, Oxygen uptake rate (mmol Cell− 1 h− 1); RCCS, Rotary cell culture system; SSE, Sum of square
errors; STR, Stirred tank reactors.
* Correspondence to: Department of Chemical Engineering, Faculty of Engineering, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
E-mail address: stai@sun.ac.za (S.L. Tai).

https://doi.org/10.1016/j.bej.2022.108794
Received 1 November 2022; Received in revised form 20 December 2022; Accepted 22 December 2022
Available online 26 December 2022
1369-703X/© 2022 Elsevier B.V. All rights reserved.
R. Sharma et al.
Nomenclature
C* Oxygen saturation concentration mmol L− 1
CL Oxygen concentration at time t mmol L− 1
C0 Initial tracer concentration mol m− 3
Cp Specific heat capacity J kg− 1 K− 1
3
Ct Uniform tracer concentration at time (t) mol m−
C∞ Final tracer concentration mol m− 3
D Vessel diameter m
d Impeller diameter m
dp Particle diameter m
ds Impeller shaft diameter m
h95 Time taken to reach the 95% homogeneity S
hL,d Height of liquid covering impeller m
kL a Mass transfer coefficient h− 1
L Vessel length m
n Impeller speed rps
Nvs Superficial gas velocity air flow number -
NQ Circulation flow number [Q/nd3] -
NP Power number [P/ρLn3d5] -
NRe Reynolds number [ρLnd2/μ] -
below which respiration becomes growth limiting. This oxygen transfer
rate is met by the product of the volumetric oxygen transfer coefficient
kL a and the oxygen driving force. To meet the demand of oxygen,
different combinations of aeration and agitation can be implemented to
fit into the operating window in which excessive hydrodynamic shear,
bubble damage and foam formation are avoided while the required OTR
is met [6]. Many reactor innovations have been developed to provide
sufficient oxygen supply to the cells through microsparger and macro­
sparger, microporous silicone tube, aeration through the head space
(surface aeration), and by orbital shaking. Where air is used as the
sparged gas, the aeration and agitation configuration determine the
mass transfer potential of the bioreactor and, consequently, the shear
stress generated by this combination sets the basis for scale up consid­
erations [6].
A range of bioreactor designs have been explored for the growth of
particular cell systems based on their mode of propagation of cells. The
response to hydrodynamic shear and other physiological conditions
differs across cell lines. Apart from the conventionally aerated stirred
tank, many bioreactor designs rely on the surface aeration to minimize
the shear stress and heterogeneity. Bioreactor designs based on new
technologies such as the wave induced disposable bioreactor, shaking
bioreactor, rotatory cell culture system (RCCS), CelCradle system, dy­
namic membrane aeration technology (DMA), traveling wave biore­
actor, and tumbling bioreactor have been tested at laboratory scale and
some at commercial scale [8].
In this research, a surface-aerated horizontal tubular bioreactor
(HTB) fitted with a spiral impeller was designed to maximize the surface
aeration through maximizing surface renewal with a large surface area
on which thin liquid films form and a low liquid height and aspect ratio
through which the surface area to volume ratio is maximized for effi­
cient mass and heat transfer, minimum hydrostatic pressure from a
horizontal configuration, and efficient energy input with a magnetically
driven impeller [9]. The use of a spiral impeller in the horizontal tubular
bioreactor is unique, although researchers have used a static helical
track in a shaken bioreactor [10] and in a vertical vessel [11], but this
has not been reported yet as an impeller in a horizontal vessel for cell
culturing. The HTB was therefore fabricated, commissioned, and the
abiotic characterization in terms of engineering parameters was assessed
for its applicability for suspended and adherent (microcarrier)
mammalian cell culture in this paper.
2
Biochemical Engineering Journal 191 (2023) 108794
Njs Minimum agitation speed s− 1
P Power input W
Q Volumetric bulk fluid pumping rate m3 s− 1
S Zwietering impeller parameter -
Si Impeller pitch m
Tf Temperature of the fluid K
T∞ Temperature of the surrounding K
UA Overall heat transfer coefficient W K− 1
Vs Superficial gas velocity ms− 1
W Width of impeller blade m
Х Solids loading (weight %) -
Greek letters
δ Diffusivity of oxygen in water m2 s− 1
θ Helix angle Degree/Rad
θm Mixing time to achieve homogeneity at H(t)= 0.95 s
μ Fluid viscosity kg m− 1 s− 1
ν Kinematic viscosity m2 s− 1
ρ Density of the fluid kg m− 3
ρL Liquid density kg m− 3
3
Δρ Difference between particle and liquid density kg m−
2. Materials and method
2.1. Bioreactor design and fabrication
The 5-liter HTB with spiral impeller was designed at Centre for
Bioprocess Engineering Research (CeBER) at the University of Cape
Town (UCT), South Africa and the fabrication of the HTB was out­
sourced to GlassChem, Stellenbosch. The design of the spiral impeller in
a horizontal tubular vessel was inspired by the work of Tsui and Hu [11]
where a spiral impeller with a draft tube in a stirred tank bioreactor was
used. The prototype was designed using the basic aspect ratio of length /
diameter (L/D ratio) of the vessel. Different combinations of aspect ra­
tios (L/D) from 1:1 to 5:1 were calculated to design the impeller diam­
eter, shaft diameter, length of the impeller, pitch, height of the impeller
blade, clearance, and total volume. Details of the design and subsequent
engineering parameter investigations can be found at doi: 10.25375/uct
.20359167.
The HTB consists of a glass column of 500 mm in length (L). The
diameter (D) and height of the horizontal vessel (H) are D=H=L/4.6.
The diameter of the impeller (d) is d=D/2.2 and the shaft diameter ds
= 0.2d. The clearance (C) of the impeller is C= 0.05 d and the length of
the impeller (h) is L/1.235. The pitch (Si) of the spiral impeller is 29 mm
with a helix angle (θ) of 17 degree. The width of the impeller blade (W)
was fixed at (d-ds)/2 and the thickness of the blade at 2 mm. The pro­
totype of the HTB is illustrated in Fig. 1.
The bioreactor has a magnetic-coupled motor drive on one side
where the base of the tube reactor is made of glass and ports for addi­
tions of liquid and gas, sampling port, heating circuit and probes posi­
tioned in the stainless-steel side plate at the other end of the reactor
tube. The bioreactor is equipped with a pH probe (Metler Toledo P/N
SAP 59903230), dissolved oxygen probe (Mettler Toledo Ingold) and a
temperature sensor (PT-100). A circulatory water heater is connected to
the bioreactor for the control of the process temperature. The regulation
of gas flow is controlled by rotameters. A bioprocess controller was
assembled with pH, temperature, impeller stirring speed and gas flow
control. The biocontroller was equipped with a mass flow controller
(MFC) with solenoid valves to maintain air saturation and CO2 gas for
pH maintenance. Data acquisition software (Terminal IP) was used to
log the real-time process data into a csv document format. The schematic
representation of the HTB is illustrated in Fig. 2.
The materials used in the fabrication of the bioreactor are
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

Fig. 1. Illustration of horizontal reactor vessel with (a) Zoomed-in magnetically coupled motor drive (b) Central glass reactor vessel with spiral impeller and heating
finger and (c) side plate for addition ports.

Fig. 2. (A) Schematic diagram of the horizontal tubular bioreactor. 1. PC with Terminal program for data logging 2. Bio-controller 3. Conductivity meter and probe
4. Magnetically coupled motor drive 5. Dissolved oxygen probe 6. pH probe 7. Temperature probe 8. Spiral impeller 9. Heating finger 10. Thermostat 11. Horizontal
tubular glass reactor vessel 12. Compressed air, 13. Air/N2/CO2 supply, 14. Rotameters. (B) Side plate schematics. 15. Medium addition 16. Seed addition 17.
Primary and secondary feed addition 18. Sampling port 19. Harvest port 20. Straight heating finger.

autoclavable and inert (SS316, borosilicate glass). The sizes and posi­ in the bioreactor and calculated as follows:
tions of the ports are shown in Table 1.
h95 = (Ct − C0 )/(C∞ − C0 ) (1)

2.2. Mixing time where C0 is the initial concentration of the tracer, Ct is the uniform tracer
concentration at time (t) and C∞ is the final tracer concentration [13].
Mixing times were calculated using the conductivity tracer method
described by Coulson et al. [12]. An aliquot of 1 mL L− 1 of a 5.0 M so­ 2.2.1. Mixing time models for HTB
dium chloride (NaCl) tracer was added at each impeller immersion ratio The HTB mixing time models were modified from Tsui and Hu [11]
and impeller speed. The conductivity profile was monitored using a by incorporating the impeller immersion ratio and impeller speed, as
AZ-86555 bench-top multi-meter (AZ Instruments). The change in con­ described in Eq. (2).
ductivity was logged to computer-aided software (Handheld Version ( )( )3 ( )b
3.0) in mS cm− 1 at one second intervals. The time taken to reach the
π L D hL,d
θm,model1 = a (2)
95% homogeneity, h95 , was used as an indicator of the extent of mixing n NQ D d d

where NQ is the circulation flow number [-], n the impeller rotation

Table 1
speed [s− 1], L the vessel length [m], D the vessel diameter [m], d the
Description of ports, their standard sizes and the location on the vessel.
impeller diameter [m], hL,d the liquid height covering the impeller
Port Size Position on the vessel diameter [m], and a and b the variable parameters. Model 1 was further
Dissolved Oxygen M18×1.5 Corner of the side of the glass wall near modified to include additional parameters as shown in Eq. (3).
sensor magnetic coupling (Fig. 2, part no. 5)
( )( )3 [( )b ( ) ]
pH Probe M18×1.5 Corner of the side of the glass wall near π L D hL,d hL,d
magnetic coupling (Fig. 2, part no. 6) θm,model2 = a − c +d (3)
n NQ D d d d
Temperature sensor PT- 10 mm Detachable part for the sensor on the vessel
100 OD with no media contact (Fig. 2, part no. 7)
The Buckingham π-Method was used to develop model 3 as a func­
Straight Heating Finger M18×1.5 At the bottom of the vessel (Fig. 2, part no. 9
& 20) tion of dimensionless groups of power number Np, Reynolds number
Medium addition 10 mm Top of the side plate (Fig. 2, part no. 15) NRe, and circulation number NQ as shown in Eq. (4).
OD ( )α
Seed Addition 10 mm Top of the side plate (Fig. 2, part no. 16) hL,d
θm,model3 = K (NP )β (NRe )γ (NQ )δ (4)
OD d
Primary and secondary 10 mm Top of the side plate (Fig. 2, part no. 17)
feed addition OD The value of a, b, c, d, K[s], α, β, γ, δ were calculated by least square
Sampling Port 6 mm OD Bottom of the side plate (Fig. 2, part no. 18) regression using Microsoft Excel’s solver function.
Harvest port 10 mm Bottom of the side plate (Fig. 2, part no. 19)
OD
Triple addition port for M18×1.5 Top of the glass vessel 2.3. Fluid flow pattern
base
Air Vent filter 10 mm Top of the glass vessel Fluid flow pattern was determined by using the phenolphthalein
OD
tracer method [14]. The experiments were conducted at each impeller

3
R. Sharma et al.
immersion ratio (percentage of impeller diameter submerged in liquid)
of 4% (1 L), 42% (2 L), 79% (3 L) and fully immersed impeller of 121%
(4 L) operated at impeller speeds of 100, 200, 300, 400, and 500 rpm.
Using 2 mL of phenolphthalein (Merck – 107227) per 1 litre working
volume of deionized water in the reactor, a tracer volume of 1 mL L− 1 of
1.0 M NaOH (Merck – 1064825000) was added to the reactor as a pulse
and the color change with the acid – base reaction tracked. A Canon
EOS-600 DSLR camera was set up facing the reactor, and images of 1–2
frames per second (fps) were taken. Thereafter, 1 mL L− 1 of 1.0 M HCl
(32%) (Merck – 1003192500) was added to turn the pink solution to
colorless.
2.4. Minimum agitation speed
The minimum agitation speed (Njs ) for suspension was calculated at
different impeller immersion ratios by varying the solid suspension by
mass. The bioreactor was operated at impeller immersion of 42% (2 L),
79% (3 L), and fully immersed (4 L) with solid loading (beads) of 0.25%,
0.50%, 0.75% and 1.00% by mass (g/g). The 4% impeller immersion
(1 L) volume was not considered due to low contact between impeller
blades and the liquid surface. The heating finger was removed to avoid
beads being trapped between the heating finger and the bottom of the
reactor. The bioreactor was filled with deionized water and loaded with
uniform sized (200 ± 50 µM @ 1.013 g/cm3 density) sodium alginate
beads to mimic microcarriers [15] stained with 10 µg mL− 1
Rhodamine-B dye (Sigma - Cat. No. R6626 – 25 G). The impeller speed
was initially set to 100 rpm and increased in small incremental steps
(1 rpm sensitivity/accuracy). The bioreactor was observed visually
using the criterion presented by Zwietering [16] that the minimum
agitation speed was achieved when no beads settled at the bottom of the
vessel for at least 1 s.
2.4.1. Minimum agitation speed models
The Zwietering [16] model carried out on vertical stirred tank re­
actors with different geometries was used as a basis to develop the
minimum agitation speed model for the HTB and is shown in Eq. (5).
( )0.45
S • ν0.1 • d0.2 g•Δρ
• χ 0.13
p •
ρL
Njs,o = 0.85
(5)
d Both temperature measurements were logged onto a computer system
where Njs,o is the original modeled minimum agitation speed [rps], S the
measured Zwietering dimensionless parameter [-], v is the kinematic
viscosity [m2 s− 1], dp the particle diameter [m], g the acceleration of
gravity [m s− 2], Δρ the difference between the particle and liquid den­
sity [kg m− 3], ρL the liquid density [kg m− 3], χ the solid loading fraction
[-], and d the diameter of the impeller [m]. The value of S was calculated
for the HTB for each impeller immersion ratio by measuring the mini­
mum impeller speed (Njs ) experimentally at each solid loading and using
Eq. (5). The modified HTB Zwietering model was then determined by
recalculating the coefficients K, a, b, c, d, and e by least squares
regression to minimize the sum of squares error as defined by Eq. (6).
( )c
SK • νa • dbp • g•Δ ρ dT
ρL • χd
Njs,m =
d e
2.5. Oxygen mass transfer efficiency
2.5.1. Determining the volumetric oxygen mass transfer coefficient
Static gassing-out method was used to determine the volumetric
oxygen mass transfer coefficient (kL a) in the HTB [17–19]. Dissolved
oxygen was removed by injecting nitrogen at 0.5 L min− 1 through head
space. The bioreactor was operated at 42% (2 L) and 79% (3 L) volumes
with impeller speeds of 100, 200, 300, 400 and 500 rpm. Compressed air
was used to fill the headspace at 0.2, 0.3, 0.4, and 0.5 LPM. A Mettler
Toledo polarographic oxygen sensor (InPro6850i/12/120) was used to
Biochemical Engineering Journal 191 (2023) 108794
track dissolved oxygen concentration with time at 1 min intervals, with
data logged on the in-house built software “Terminal IP”. Measurements
were carried out in duplicate for each condition. The kL a at each oper­
ating condition was determined using Eq. (7).
dCL /dt = − kL a(C* − CL ) (7)
where C* is the saturation concentration of dissolved oxygen, and CL is
the oxygen concentration at time t [20–22].
2.5.2. Oxygen mass transfer models
kL a was hypothesized to be function of the following parameters
( )
kL a = f hL,d , d, n, P, ρ, μ, vs , Q, D, L, Si , δ
where, hL,d is a height of liquid [m], d is impeller diameter [m],n
impeller speed [rps], P power input [W], ρ density of the fluid [kg m3], μ
fluid viscosity [kg m− 1 s− 1], vs superficial velocity [m s− 1], and Q
volumetric bulk fluid pumping rate [m3 s− 1], Parameters D and L
(diameter and length of the vessel [m]), Si is the impeller pitch [m], and
δ is the diffusivity of oxygen in water [m2 s− 1] were fixed parameters
and were included in the pre-exponential constant K1 . To investigate the
inter-dependence of the power input, Reynolds numbers, flow number
and impeller clearance, the dimensional analysis was done using the
Buckingham π-Method, which provided the following non-linear
expression for kL a (Eq. (8)).
( )α (
hL,d P
)β ( )γ ( )
ρnd2 ( vS )δ Q ε
kL a(h− 1 ) = K1 (8)
d ρn3 d5 μ nd nd 3
The values of these exponents were determined by non-linear least-
squares regression using the least squares function in Scilab.
2.6. Power utilization
The power consumption of the HTB was determined through the
temperature method [23,24]. The bioreactor was insulated thoroughly
by wrapping it with sheets of cotton wool. Two temperature sensors
were used, one placed inside the bioreactor submerged in liquid and one
outside of the bioreactor to measure the temperature of the surrounding.
“Terminal IP”.
Impeller immersion ratios of 42% (2 L), 79% (3 L) and the fully
immersed impeller (4 L) were used to determine the power consumption
at different impeller speeds ranging from 100 rpm to 500 rpm with the
increments of 100 rpm. The water in the bioreactor was heated to
35.5 ◦ C and the temperatures inside and outside of the bioreactor were
continuously logged until both temperatures reached an equilibrium
temperature (steady-state). Raval et al. [24] described the energy bal­
ance as shown in Eq. (9).
dTf ( )
− mCp = UA Tf − T∞ − P (9)
dt
where − mCp dtf represents the cooling rate of the heated liquid in the
(6)
bioreactor, m (kg) represent the mass of the fluid, Cp indicated the
dT
specific heat capacity (J kg− 1 K− 1) and dtf denoted the rate of change of
( )
temperature. UA Tf − T∞ represents the heat loss to the environment,
which is represented by overall heat transfer coefficient UA (W K− 1) and
P (W) denotes the power dissipation to the liquid. Two temperatures Tf
and T∞ (K) are the temperature of the fluid inside the bioreactor and the
surroundings (outside the insulating cotton sheets) respectively. Power
(P) was zero when the stirrer was kept off resulting in Eq. (10).
dTf ( )
− mCp = UA Tf − T∞ (10)
dt
Eq. (10) was solved for the overall heat transfer coefficient (U) and
4
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

area of heat loss (A). Thereafter, experiments were set up with different HTB followed the general trend where the increase in agitation speeds
stirring speeds to calculate the value of P by solving Eq. (9). P was solved resulted in a non-linear decrease of mixing time at all fill volumes.
by minimizing the sum of square errors (SSE) using the Microsoft Excel’s Mixing was the less effective at 4% impeller immersion and took a long
Solver function. time because of the low impeller-fluid interaction. The experimental
Power number, Np and flow number NQ were predicted for the HTB value at 100 rpm with 4% immersion was not shown as the mixing time
using the model developed for a vertical screw impeller Eq. (11). was long (>600 s – data not shown) and the standard deviations were
large. Fig. 3 shows that mixing time decreases dramatically for all
Np NRe
= 600 (11) impeller immersions on increasing impeller speed from 100 to 200 rpm
NQ
and gradually becomes steady as it approached 500 rpm.
Using Eq. (11), the Reynolds numbers were calculated for each Impeller immersion has a significant effect on mixing time through a
impeller speed, and power numbers NQ and Np were predicted from non-linear relationship. There is a significant decrease in mixing time on
estimated circulation number through mixing time. increasing impeller immersion from 4% to 42%, as the impeller has
more contact with and is able to transfer more energy to the liquid
2.7. Statistical tools and analysis volume, moving fluid elements throughout the vessel. However, a
change from 42% to 79% impeller immersion has less impact on mixing
A simple percentage error was calculated for all predicted model time. The geometry of the vessel may also explain this deviation; from
values with respect to the experimental values. This was done using Eq. 42% to 79%, the liquid is at the widest point in the cylindrical reactor,
(12). which means that a change in liquid volume did not lead to a significant
change in vessel liquid height. This may be further compounded by the
%error =
|Experimentalvalue − Modelvalue|
× 100 (12) fact that with larger liquid volumes, the backflow is negated, reducing
Experimentalvalue circulation and increasing mixing time.
The coefficient of determination, R2-value was determined using the When increasing the impeller immersion to 121% (4 L), the mixing
following Eq. (13) for the various comparison of model fit to the time increased compared to the 2 and 3 L fill volume. A fully submerged
experimental value. impeller without mixing at the air/liquid interface seemed to be less
beneficial, possibly as a result of increased energy requirements against
R2 =
SSE
(13) the increased viscous and drag forces in the liquid, hence making less
SST energy available for liquid mixing. This theory also supports the para­
bolic shaped mixing time profiles when increasing from 100 to 500 rpm.
where, SSE is the sum of squared errors and SST is the total sum of
At lower rotation speeds, most energy is used in counteracting the liquid
squares.
drag forces, but as the rotation speed increases, the required energy to
The residuals are the errors between the experimental and modeled
overcome drag forces becomes constant, and the energy supplied into
values, calculated by Eq. (14). These are used to form the residual plots.
the fluid can be used for mixing activities. It is also postulated that one of
Residual = Experimentalvalue − modelledvalue (14) the reasons the mixing time increased at 4 L was that the axial flow
generated by the impeller was enhanced by full submergence, which
A two-way analysis of variance (ANOVA) was carried out on the
negated the backflow generated by the pressure gradient leading to a
mixing time and kL a studies using Microsoft Excel’s ANOVA with the
reduced overall circulation flow and longer mixing time. Therefore,
replication data analysis tool at a confidence level of 95% (α = 0.05).
there is a region of desirable operating conditions between 42% and
79% impeller immersion, where the mixing efficiency is at its highest
3. Results and discussion
and also where mixing speed affects mixing time less, showing operation
stability.
3.1. Mixing time
3.1.1. Comparison of mixing models
The mixing time was determined at different fill volumes or impeller
The three models proposed with Eqs. (2)–(4) were fitted with the
immersion ratios and different impeller speeds in the HTB in the pres­
experimental data obtained. The following parameters were obtained
ence of tracer (5 M NaCl) as shown in Fig. 3. The mixing time profile of
and listed in Table 2.
Comparison of the mixing models were carried out numerically using
200 percentage error, R2-values and residuals plots for the best fit model to
180 the experimental data. Table 3 summarizes the percentage errors and R2
values associated with each model with respect to the impeller immer­
160
sion and impeller speed together with an overall error.
140 Model 2 appears to be the best fit as it showed less error than other
Mixing time (s)

120 models with a strong R2 value score across the conditions fitted. Model
2, much like Model 1 which incorporated the vessel length, vessel and
100
impeller diameter, and impeller immersion ratio but with an extended c
80 and d parameter and function showed flexibility in accommodating the
60 changes within the ranges tested. When impeller speed was plotted
against mixing time (Fig. 3), the impeller speed showed an inverse
40
exponent greater than 1 towards mixing time, while when impeller
20
0
Table 2
0 100 200 300 400 500 600
Impeller speed (rpm) List of parameters for mixing models.
Mixing model Parameters
Fig. 3. Relationship between mixing time and impeller speed at different fill
volumes. ▴denotes 4% immersion (1 L), ■ denotes 42% immersion (2 L), • θm,model1 a = 1.00, b = 0.55
θm,model2 a = 1.00, b = 0.98, c = 0.45, d = 0.19
denotes 79% immersion (3 L), and ◆ denotes 121% fully immersed (4 L). Error
θm,model3 K = 160,945, α = 0.51, β = 0.06, γ = − 0.89 δ = − 1.19
bars show the standard deviation of n = 3 replicate samples.

5
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

Table 3 more favorable.

Percentage error and Coefficient of variance (R2 values) of each mixing model
with respect to impeller speed and impeller immersion. The acceptable 3.2. Fluid flow patterns in the HTB
threshold for error should be less than 15%, while that for the R2-value is above
0.8 [25].
The effects of impeller immersion on the fluid flow pattern were
Parameter Percentage error (%) R2 values investigated as the spiral movement was hypothesized to move the fluid
Impeller θm,model1 θm,model2 θm,model3 θm,model1 θm,model2 θm,model3
across the horizontal tube in a counter-current flow in order to achieve
speed
(rpm) good mixing. For visualization, three images of decolourization of
100 34 <1 5 0.92 1.00 1.00 phenolphthalein dye over time at specific fill volumes are shown in
200 34 <1 7 0.83 1.00 0.99 Fig. 5 at an impeller speed of 300 rpm.
300 34 <1 5 0.87 1.00 1.00 The last image (right-hand side) shows a slight pink streak which
400 34 <1 7 0.82 1.00 0.99
500 34 <1 10 0.82 1.00 0.99
indicate the moment before complete decolourization. The mixing time
for 95% homogeneity at 4% impeller immersion was the longest for all
Impeller Percentage error (%) R2 values the volumes tested. The reason for this is as indicated with the mixing
immersion time studies is because of the low impeller immersion ratio, resulting in
(%)
low axial flow motion of fluid or that the blades are unable to create a
4 19 <1 2 0.93 1.00 1.00
42 47 <1 20 0.00 1.00 0.89 push in the fluid along the length of the vessel. Similarly, the decolou­
79 39 1.5 4 0.52 1.00 1.00 rization for 2 L, 3 L and 4 L showed corresponding behaviors seen with
121 32 <1 3 0.61 1.00 0.99 the mixing time conductivity studies.
Overall 34 <1 7 0.70 1.00 0.98 On zooming into the region near the heating element, decolourizing
happened faster than the area without the element and generally faster
immersion was plotted against mixing time (data not shown), a mini­ at the bottom half as compared to the region near to the gas-liquid
mum mixing time was obtained, suggesting that the relationship can be interface. The heating element was observed to act like a baffle,
modeled with a second order polynomial equation. Model 3, modeled enhancing the mixing homogeneity in that region of the reactor, aided
with dimensionless numbers showed promising fitness to the experi­ by turbulent eddies between the impeller blades and near the tip as
mental data, however, the overall percentage error was slightly above shown in Fig. 6 with black circles. Higher impeller speeds increased
Model 2, indicating rigidity and therefore required improvements to the homogeneity in the ‘un-baffled’ region, but the relative improvement
model. decreased with increasing impeller speed. If a two-compartment mixing
To solidify the findings above, the residual plots for mixing time on model is applied, consisting of two tanks in series, the region which
impeller immersion and speed effects were plotted (Fig. 4). In agreement contained the heating element would have a shorter mixing time than
with the percentage error and R2-values, Model 2 showed less scatter. the region without the heating element.
Models 1 and 2 were developed from a model proposed by Tsui and Hu With the increase in impeller agitation, decolourization time de­
[11], which was used for a vertical screw impeller system. Tsui and Hu’s creases, whilst the flow of fluid becomes more turbulent. This turbulence
modified Model 2 is therefore accepted for its applicability for the HTB at high speed improves mixing in the system, thus reducing the con­
system. Model 3 was derived using the Buckingham π-method and is centration gradient of the tracer more quickly. The agitation speed at
widely accepted as a robust way to develop empirical models, though 300 rpm or above, showed surface vortices forming and these grow in
the core variable selection procedure may sometimes be arbitrary. While numbers as the impeller agitation increases (data not shown). At
both Model 2 and 3 come with uncertainties, the Model 2 derived is 500 rpm, the spiral impeller entrained air bubbles from the headspace
through the impeller blade into the bulk liquid which increased the

Fig. 4. Residual plots for all the mixing models with respect to impeller immersion and impeller speed. ○ denotes 100 rpm, ▢ denotes 200 rpm Ж denotes 300 rpm, Δ
denotes 400 rpm, ⋄ denotes 500 rpm. ▴ denotes 4% immersion (1 L), ■ denotes 42% immersion (2 L), • denotes 79% immersion (3 L), and ◆ denotes 121% fully
immersed (4 L).

6
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

4%
immersion
at 300 rpm
(1 L) 383 s 391s 398s
42 %
immersion
at 300 rpm
(2 L) 38s 43s 51s
79 %
immersion
at 300 rpm
(3 L) 32s 39s 48s
Fully
immersed
at 300 rpm
(4 L) 54s 65s 71s

Fig. 5. Schematic progression of homogeneity attained at different impeller immersion at 300 rpm. Time is seconds indicates the moment before decolourization
from the start of the respective experiments.

suspension Njs are reported in Table 4. As the solid loading percentage

was increased, the minimum agitation speeds (rpm) increased accord­
ingly. At 2 L and 3 L fill volumes, no significant difference were seen
when the solid load percentages were between 0.25% and 0.5%, but the
divergence was bigger at solid loadings of 0.75% and 1.00%, aligning
with the fluid flow patterns examined at these volumes. At 4 L, the
divergence across the solid loading percentages were much lower then 2
and 3 L fill volumes.
The value of S was determined experimentally using Eq. (5) by
measuring the minimum impeller speed, Njs to meet the off-bottom
criteria of the beads at each impeller speed and solid loading percent­
age for each fill volume (Table 4). Thereafter, the modeled (Njs,m ) Eq. (6)
with the experimentally determined S value were used and the co­
efficients were calculated by least squares regression to minimize the
sum of squares error resulting in Eq. (15).
Fig. 6. Turbulent eddies formed (circled) between impeller blades above the
( )0.092
heating finger. The turbulent flow eddies formed are normal to the ρ
S0.965 • ν0.048 • d0.046 • g•Δ • χ 0.32
viewing plane. Njs,m =
p ρL
(15)
d0.10
oxygen transfer into the liquid but would lead to mammalian cell death From the experimental data in Table 4, the increment in solid loading
due to bubble bursting [7,26]. at 42% (2 L) and at fully immersed impeller at 4 L showed an expo­
nential increase in the value of Njs , whereas for the 79% immersion (3 L),
3.3. Minimum agitation speed the increase was not proportional. This indicates that off-bottom clear­
ance is much better for all the solid loadings at 3 L volume than at 2 L
The experimental minimum critical agitation speeds for solids and 4 L. However, at 0.25% and 0.50% of solid loadings, there was small
difference in experimental Njs between 2 L and 3 L, leading to a
maximum value in experimental Njs at 0.75%. This trend could not be
Table 4
modeled and predicted effectively as at solid loading of 0.75% and
Experimental (Njs), original Zwietering model (Njs,o), and modified Zwietering
model (Njs,m) minimum agitation speeds in rpm for different fill volumes and 1.00% the plateauing ofNjs was not seen and the model was unable to
solid loading percentage. S coefficient reported for each fill volume. Standard follow this trend. Many of the minimum suspension speeds lie above
deviation is the deviation found across averaging S value over the four different 500 rpm, especially at 0.50%, 0.75% and 1.00% solids loading. From the
solid loading percentages of 0.25%, 0.50%, 0.75% and 1.00% within the same results gathered in other investigations, the optimum impeller immer­
fill volume. The immersion level of the impeller for the different fill volumes are sion operating point is between 42% and 79%, where Njs is typically
2 L (42% submerged), 3 L (79% submerged), 4 L (fully submerged at 121%). above 500 rpm. This is problematic, as higher impeller speeds lead to
Fill volume χ (solid load percentage) S coefficient higher shear stress on the cells.
The S coefficient decreases with an increase in impeller immersion.
0.25 0.50 0.75 1.00
This is surprising trend, as Zwietering [16] shows that S increases lin­
2L (Njs) 450 530 670 750 33 ± 2
early with T/D on a log-log plot. There is a similar downward trend in Njs
2L (Njs,o) 535 586 618 641
2L (Njs,m) 456 569 648 711 as impeller immersion increases. This difference in trend may be
3L (Njs) 465 560 590 580 30 ± 1 attributed to the significant differences in the system under study i.e.
3L (Njs,o) 493 540 569 591 horizontal vs vertical. As impeller immersion increases, the impeller has
3L (Njs,m) 421 526 599 657 more contact with the liquid volume. This leads to more movement of
4L (Njs) 220 240 320 460 17 ± 2
4L (Njs,o) 275 301 317 330
the liquid volume, and consequently better suspension of any solids in
4L (Njs,m) 240 300 341 374 solution. This is different to vertical STRs, where the impeller is always

7
R. Sharma et al.
submerged in the liquid volume.
When the modified Njs,m was compared to the original Zwietering
equation developed for STRs (Njs,o), the percentage error, R2-values and
residual plots suggest that the modified Njs,m which takes into account
the horizontal nature of the reactor design, correlates better with the
experimental data. The tabulated information given in Table 5 dis­
playing the percentage error and the coefficient of determination (R2-
values) while Fig. 7 gives the residual plots.
The R2 values associated with fitting the original Zwietering equa­
tion range between 0.32 and 0.88, illustrating its poor fit. This is in
contract to the modified equation resulting in R2 values in the range
0.93–0.99. The improved fit of the modified equation is further sup­
ported by the reduced error associated with it over the original equation
i.e 4–15 (overall 9) and 4–20 (overall 12) respectively. A particular R2
value for the 79% (3 L fill volume) Njs,m model resulted in a very low
score. The large deviation occurring at 3 L is inline with the unexpected
plateauing of the minimum agitation speed experimentally measured
across the different solids load percentages as previously described. The
residual plots of impeller immersion and Njs with solid loading indicate
the improvement seen with the modified equation Njs,m, with the plot
showing residuals in a tighter band than when using the original equa­
tion Njs,o which was developed for a STR.
3.4. Mass transfer
From the mixing studies, it was found that 1 L and 4 L volume were
not suitable conditions under which to operate the HTB, therefore, the
mass transfer studies were conducted at 2 L (42%) and 3 L (79%) vol­
umes (impeller immersion) only. The experimental data shown in Fig. 8
indicate that the increase in impeller speed and airflow rates increase
the kL a. The difference in the kL a was not prominent at 42% and 79%
impeller immersion, this could be due to limited increase in the liquid
height when the fill volume increased from 2 L to 3 L. This is also in line
with the similar mixing times reported at these volumes. The range of
the volumetric mass transfer coefficient exhibited by the HTB at all the
agitation speeds fell between 5 and 16 h− 1 which is in accordance with
the widely reported range for mass transfer of 1–15 h− 1 for mammalian
cell culture in a STR [2].
The mass transfer efficiency in a horizontal bioreactor primarily
depends on the agitation speed, impeller immersion ratio, airflow rate
and resultant surface renewal rates. The use of spiral impeller with
multiple blades along the length of the vessel provides a large surface
area to volume ratio for mass and heat transfer. The spiral design of the
impeller also imparts uniform distribution of energy to the fluid due to
high fluid movement between the blades across the length of the vessel.
This is possible because of the multiple energy distribution points
available across the length in the form of impeller blades, this would
Table 5
Percentage error (average of 2, 3 and 4 L) and Coefficient of variance (R2 values -
average of 0.25%, 0.50%, 0.75% and 1.00% solid loading) for the standard
Zwietering equation for STR (Njs) and modified Zwietering equation for HTB
(Njs,m) with respect to varying solids loading and impeller immersion. The
acceptable threshold for error should be less than 15%, while that for the R2-
value is above 0.8 [25].
Solids loading (%) Error (%) R2
Njs,o Njs,m Njs,o
0.25 17 7 0.71
0.50 13 13 0.88
0.75 4 4 0.95
1.00 15 12 0.32
Impeller immersion (%)
42 13 4 0.54
79 4 8 0.82
Fully immersed 20 15 0.33
Overall 12 9 0.65
Biochemical Engineering Journal 191 (2023) 108794
enhance gas hold-up volume due to this redistribution and result in
increased mass transfer efficiency [27]. In addition, the bubble
entrainment seen at higher rotational speeds aids oxygen mass transfer
into the liquid phase, as identified previously in the fluid flow pattern
studies.
When modeling for kL a, the values of the exponents in Eq. (8) were
determined by the method of non-linear least-squares regression and
were substituted into Eq. (16) and this equation was proposed for a
surface aerated horizontal tubular bioreactor with screw impeller, under
abiotic conditions.
( )0.08 ( )− 0.90 ( )− 0.03 ( ( )0.79
hL,d P ρnd2 vS )0.48 Q
kL a(h− 1 ) = 0.36
d ρn3 d5 μ nd nd3
(16)
The model given in Eq. (16) shows a very low dependence of kLa on
impeller immersion (1st term) with an exponent of 0.08. This is believed
to be due to the limited impeller immersion range tested of 42% at 2 L
volume and 79% at 3 L. A large increase in impeller immersion repre­
sents a small increase in volume and liquid height. The dependence on
Reynolds number (3rd term) was found to have an exponent of − 0.03.
Circulation flow number (5th term), NQ, had the second largest depen­
dence after power number (2nd term), with exponents of 0.79 and
− 0.90 respectively. This indicates that convective mass transfer is an
important influence on kLa. The amount of convective mass transfer in a
system strongly influences concentration gradients, and the dependence
of kLa on convective transport phenomena indicates that kLa is also
dependent on concentration gradients. The exponent of the superficial
gas velocity air flow number, NVS (4th term), was found to be 0.48
indicating a substantial role of air federate in the determination of kL a.
Eq. (16) was further simplified by substituting the value of the fixed
impeller diameter (d = 0.050m), and assuming temperature of 37 ◦ C for
constant water density, ρ and viscosity, μ leading to Eq. (17).
hL,d 0.08 vS 0.48 Q0.79 n1.41
kL a(h− 1 ) = 1.29 (17)
P0.90
The largest dependence of kL a was on impeller rotational speed with
an exponent of 1.41. These dependencies on the speed of the impeller
indicate that kL a is most strongly a function of energy dissipation.
Power, with an exponent of − 0.9 indicates that kLa is influenced about
twice as strongly by energy dissipation than by aeration, given by the
exponent of 0.48. The opposite signs here do not indicate an inverse
relationship, and the effects of impeller speed and aeration rate on kL a
are additive.
The residual plots for the kL a models at 2 and 3 litres are shown in
Fig. 9. The corresponding overall coefficient of determination for the fit
of the kL a model data to the experimental data was determined to be R2
= 0.94. The residues were more prominent in the 3 L batches with the
highest scatter seen at higher air flowrates and kL a values.
3.5. Power consumption
The power consumption profile of HTB at 42% (2 L), 79% (3 L) and
at fully immersed impeller (4 L) are shown in Fig. 10. It was observed
that with the increase in both impeller speed and impeller immersion
ratio, power consumption increases. This is a result of increased kinetic
energy input and shear imparted by the bulk fluid on the impeller due to
Njs,m the viscosity of the liquid and inherent drag forces.
0.98 When the impeller is fully immersed, the impeller experienced an
0.97 increased drag force compared to 42% and 79% immersion. Combina­
0.99 tion of Figs. 3 and 10 show that the mixing time profile is inversely
0.95
proportional to power consumption for each impeller immersion. The
0.93 main purpose of the impeller design is to reduce mechanical shear and
0.08 mixing time while improving mass transfer efficiency. This could be
0.67 achieved by installing specific impellers that can provide high flow
0.80 NQ
number(NQ ) and low power number (Np) to achieve a higher Np ratio
8
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

Njs,o Njs,m
Residual minimum agitation speed

150 150

100 100

50 50
(rpm)

0 0

-50 -50

-100 -100
2L 3L 4L 2L 3L 4L

Fill Volume (L)/ Impeller immersion (%)

Fig. 7. Residual plot of Zwietering equations with respect to fill volume/impeller immersion from the original equation Njs,o Eq. (5) and modified Njs,m Eq. (15) for
HTB. ▴denotes 0.25%, ■ denotes 0.50%, • denotes 0.75%, and ◆ denotes 1.00% solid load percentages.

Fig. 8. Experimental (black dot) and modeled values (shaded gray surface) of kL a (h− 1) for (A) 42% impeller immersion (2 L volume) and (B) 79% impeller im­
mersion (3 L volume). Values are representative of independent triplicate runs. n (revolutions per second, s− 1), Q (airflow rate, l min− 1).

42 % impeller immersion (2 L) 79 % impeller immersion (3 L)

4.00 4.00
3.00 3.00
2.00 2.00
Residual kLa (h-1)

1.00 1.00
0.00 0.00
-1.00 -1.00
-2.00 -2.00
-3.00 -3.00
0.2 0.3 0.4 0.5 0.2 0.3 0.4 0.5

Air Flowrate(l min-1)

Fig. 9. Residual plots for kL a models at 2 L and 3 L with respect to air flowrates. ○ denotes 100 rpm, □ denotes 200 rpm Ж denotes 300 rpm, Δ denotes 400 rpm, ◊
denotes 500 rpm.

which favors bulk mixing at a lower shear rate and the same power input
[28].
For a specific impeller geometry at a given impeller speed, mixing
time is inversely proportional to flow number and directly proportional
to power number [29]. From Table 6, it can be concluded that as the
impeller speed increases at a specific impeller immersion (fill volume),
Reynolds number increases and power number decreases. Since NRe and
NP are a function of impeller diameter (d) and impeller speed (n), in the
context of this reactor design of 0.05 ≤ d ≤ 0.09 m and
1.7 ≤ n ≤ 8.3 rps, it was found that the increase of the impeller speed

9
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

1.4 3.6. Two way ANOVA analysis and reactor comparison

1.2
Two-way ANOVA analyses with replication were done on the mixing
1.0 times and kL a studies. For mixing studies, the significant effects of
impeller immersion and impeller speed were tested, whilst for kL a
Power (W)

0.8
0.6
0.4
0.2
0.0
200 300 400 500
Impeller speed (rpm)
Fig. 10. Power consumption profile of HTB at different volumes at various
agitation speeds. ■ denotes 42% immersion (2 L), • denotes 79% immersion
(3 L), and ◆ denotes 121% fully immersed (4 L).
had a larger effect on improving mixing times. In addition, since the HTB
spiral impeller is present across the length of the horizontal vessel,
agitating the liquid at a low impeller speed consumed similar to lower
power when compared to traditional STRs [30,31].
Relating the P/V value towards achievable kL a, and with the inten­
tion of growing cell cultures to medium density, a kL a of 12–17 h− 1 is
favorable [28]. This would mean the HTB would operate within the
higher power consumption region of 300 rpm and above, and with a
higher airflow input (Fig. 11). To achieve a higher density cell culture in
the range of 5–10 × 106 cells/mL in HTB, a higher kL aof 20–28 h− 1 is
required and in the current configuration, the HTB system cannot meet
this oxygen requirements.
studies, the airflow rate and impeller speeds were tested at the 2 and 3 L
fill volume respectively. The F-values, Fcrit and p-values for each sta­
tistical analysis is shown in Table 7.
The conclusions drawn from Table 7 show that impeller immersion
has a highly significant effect on mixing time. This result is expected as
impeller immersion affects the amount of contact between the impeller
and the liquid volume and how effectively it can move liquid elements
around the vessel. This is the mechanism by which mixing occurs and
has a direct effect on mixing time. The immersion p-value is also in many
orders of magnitude lower than the α-value (0.05) and therefore the
relationship between impeller immersion and mixing time is statistically
significant and the results from the F-test are reliable.
Similarly, impeller speed has a significant effect on mixing time.
Again, this result was expected as the impeller speed is related to the rate
at which energy is dissipated from the impeller to move the liquid vol­
ume. The faster the rate of energy dissipation, the faster fluid elements
move around the vessel, leading to a faster mixing time. However, this
effect is less significant than the immersion effect, confirming a trend
observed earlier with respect to the small change in mixing times with
impeller speed at 42% and 79% immersion. The impeller speed p-value
is also in many orders of magnitude lower than the α-value, and there­
fore the relationship between impeller speed and mixing time is statis­
tically significant and the results from the F-test reliable.
Finally, both the P-test and F-test for interaction reject the null hy­
potheses and it can be said that there is significant effect on mixing time
from the interaction between these two independent variables. This

Table 6
Predicted values of power numbers and circulation flow numbers. P/V values are not available for 100 rpm as experiments were not carried out.
Liquid volume Impeller speed [rpm] Mixing time θm [s] NRe NQ Predicted Np Predicted NQ P/V value [W m− 3]
Np

2L 100 72.0 5985 0.533 0.054 9.87 N/A

200 41.3 11,970 0.465 0.023 20.22 43.5 ± 1.4
300 27.7 17,955 0.463 0.015 30.87 104.5 ± 5.5
400 20.7 23,940 0.465 0.012 38.75 147.4 ± 13
500 20.3 29,927 0.378 0.008 47.25 239.0 ± 3
3L 100 70.7 5985 0.815 0.082 9.94 N/A
200 24.7 11,970 1.168 0.059 19.80 55.0 ± 7.0
300 17.7 17,955 0.976 0.033 29.58 130.0 ± 4.7
400 16.3 23,940 0.815 0.020 40.75 195.0 ± 21.3
500 20.3 29,927 0.705 0.014 50.36 323.3 ± 9.3

Fig. 11. Mass transfer efficiency, kL a (h− 1) vs P/V value (W m− 3) at (A) 42% impeller immersion/2 L working volume and (B) 79% impeller immersion/3 L working
volume. Δ denotes 0.2 LPM, ▢ denotes 0.3 LPM, ○ denotes 0.4 LPM, ◊ denotes 0.5 LPM (litres min− 1) air flow rate. Standard errors are representative of independent
duplicate experiments.

10
R. Sharma et al. Biochemical Engineering Journal 191 (2023) 108794

Table 7 Table 8
Two-way ANOVA analysis with replication for mixing times and kL a (2 L and Comparison of different bioreactor systems w. r. t. mixing time, mass transfer
3 L) data. efficiency and power consumption profile. N/A: Not available.
Test F- Fcrit p-value p < 0.05 F< Fcrit Type of Bioreactor Mixing Mass transfer P/V value Ref.
value time (s) (kL a) (h− 1) (W m− 3)
18
Mixing Impeller 99.04 2.84 1 × 10− Yes Yes 5 L Horizontal Tubular 16–72 5.25–16 43.5–323 This
times immersion Bioreactor (HTB) (2–3 L) work
17
Impeller 64.10 2.61 7 × 10− Yes Yes Stirred tank bioreactor 3.4–27 6.5–35 0.26–38.3 [32]
speed (Stirred Mobius Cell
− 15
Interaction 27.82 2.00 1 × 10 Yes Yes Ready 3 L bioreactor)
10
kL a (2 L) Aeration rate 57.99 3.10 5 × 10− Yes Yes Wave-induced 5–10 s 4 (10–100 L) N/A [33]
11
Impeller 45.10 2.87 1 × 10− Yes Yes (10 L)
speed 60 s
Interaction 2.32 2.28 0.046 Yes Yes (100 L)
3
kL a (3 L) Aeration rate 6.65 3.10 3 × 10− Yes Yes Oscillatory baffled 12–45 4.9–17.2 216–461 [21]
8
Impeller 28.27 2.87 6 × 10− Yes Yes photobioreactor
speed Orbitally shaken 10–30 4–30 500 [34]
Interaction 1.18 2.28 0.356 No No bioreactor (OSR) (120 rpm/
1.5 L)

means that impeller speed and impeller immersion interact with respect
to affecting mixing time; keeping one variable constant is not necessarily
the same as removing the effect of that variable on mixing time.
A Two-way ANOVA for a non-random causality between impeller
speed and kL a, aeration rate and kL a, and the interaction between the
two variables and kL a showed that the effect of impeller speed on kL a is
significant within the 95% confidence interval. It was also shown that
the effect of aeration rate on kL a is significant within the 95% confidence
interval. Increasing the aeration rate naturally delivers more oxygen to
the system, and therefore increases the mass transfer efficiency by
keeping a higher oxygen concentration driving force between the gas
and liquid interface. Similarly, as with the ANOVA analysis of mixing
time with impeller speed, increasing impeller speeds results in a higher
surface renewal in the reactor gas-liquid interface, and therefore con­
tributes to the higher driving force and surface area for mass transfer
into the liquid phase.
The third hypothesis for the ANOVA was partially confirmed. An
interaction between aeration rate and impeller speed has a significant
influence on kL a at 2 L, within the 95% confidence interval, but at 3 L
volume, the interaction does not have a significant effect on kL a in the
95% confidence interval. The P-test and F-test for these interactions
were also less significant in magnitude, which may suggest that one
variable imposes the bottleneck in mass transfer and therefore the
combination effect is less pronounced in contribution towards mass
transfer efficiency.
On characterizing the design of the HTB through its abiotic testing,
the HTB showed pros and cons. When comparing the HTB with the STR,
wave-induced, shaken, PBR and CelCradle reactors, the reactor was
comparable in mixing abilities, oxygen mass transfer and power con­
sumption (Table 8). Notably, the volumetric mass transfer coefficient
kL a, achieved similar ranges as those that were aerated via surface
renewal or without a mechanical stirrer, i.e. wave-induced, oscillatory
baffled reactors and orbitally shaken bioreactors. Stirred tank bio­
reactors remain superior in mass transfer capabilities, with combinato­
rial benefits from direct air sparging and the axial motion generated
from impeller mixing.
4. Conclusions
culturing, however this is at the expense of a high rotational impeller
speed of 500 rpm, which may be detrimental to the shear sensitive cells.
The initial concept of designing a horizontal bioreactor to reduce
hydrodynamic pressure, and therefore power utilization for mixing and
mass transfer seem to live up to its potential. Through this research, the
HTB prototype and the associated models generated have given further
insight into mixing and mass transfer in horizontal agitated reactors
compared to traditional vertical vessels. Operations could be improved
by inclusion of baffles. Further, scaling up of the vessel either in diam­
eter or length should be investigated to give further indication of scal­
ability of the horizontal bioreactor. Biotic cell culture experiments to
determine the shear forces, cell death rates, cell concentration and
viability, and product formation, would indicate the suitability of the
HTB for sustainable and economical production of biologics.
CRediT authorship contribution statement
Rajesh Sharma: Conceptualization, Design of reactor, Reactor
commissioning, Methods selection, Data collection, Data curation,
Formal analysis, Investigation, Data analysis and interpretation, Vali­
dation, Writing – original draft, Writing – review & editing. Wesley
Collair: Investigation, Validation, Writing – original draft, Writing –
review & editing. Aqeel Williams: Investigation, Validation, Writing –
original draft, Writing – review & editing. Susan T.L. Harrison:
Conceptualization, Funding acquisition, Design of reactor, Methods se­
lection, Data analysis and interpretation, Supervision, Writing – review
& editing. Siew L. Tai: Conceptualization, Project administration,
Funding acquisition, Resources, Software, Supervision, Design of
reactor, Methods selection, Data analysis and interpretation, Writing –
review & editing.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Siew Leng Tai reports financial support was provided by National
Research Foundation.

The HTB, due to the horizontal nature of the design, was found to be Data availability
highly sensitive towards the liquid fill volume, which dictated the
impeller immersion percentages. When the volumes were low (1 L), I have shared a link to the datasets in the manuscript submission step.
there was insufficient mixing or motion in the fluid to keep the solids in
suspension. When the fill volume was at 80% (4 L), this discouraged
Acknowledgments
bulk flow mixing and counter-current fluid movement, and with the
impeller fully submerged, lacked bubble entrainment when compared to
The authors would like to thank the National Research Foundation
2–3 L fill volumes with partially submerged rotating impellers. The kLa
(NRF), South Africa for their financial support through the CSUR grant
recorded a maximum of 16 h− 1, which can sustain mid density cell
(UID90305) as well as through the SARChI in Bioprocess Engineering

11
R. Sharma et al.
(UID64778). Further, we would also acknowledge the guidance received
from members of the Centre for Bioprocess Engineering Research
(CeBER), Department of Chemical Engineering, University of Cape
Town, South Africa.
References
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