Journal of Cleaner Production 230 (2019) 1074e1084

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Journal of Cleaner Production

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

Process optimization in a stirred tank bioreactor based on CFD-Taguchi

method: A case study
Lei Shu a, Mingjin Yang a, Hang Zhao a, Tianfu Li b, Ling Yang a, Xiang Zou b, *, Yunwu Li a, **
a
National and Local Joint Engineering Laboratory of Intelligent Transmission and Control Technology, College of Engineering and Technology, Southwest
University, Chongqing, 400715, PR China
b
College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Cleaner aerobic fermentation with high efficiency is of great significance to microbial production. In an
Received 27 January 2019 aerobic fermentation process, gas holdup and power consumption are main indexes for its performance
Received in revised form evaluation. Based on CFD-Taguchi method, effects of control factors of impeller type, gas flow rate and
28 April 2019
agitation speed on gas holdup and power consumption were studied, by taking pullulan fermentation of
Accepted 8 May 2019
Available online 17 May 2019
Aureobasidium pullulans in a stirred tank bioreactor as a case study. Innovatively, three representative
viscosity stages and their corresponding viscosities were defined according to viscosity change during
fermentation, so as to quantitatively optimize the fermentation process. By range analysis and analysis of
Keywords:
Aureobasidium pullulans
variance, effects of control factors and their significant levels on fermentation performance were ob-
Gas holdup tained so as to optimize the fermentation system. All control factors were significant at low viscosity
Power consumption stage, and insignificant at stages of medium and high viscosity except for gas flow rate at high viscosity
Stirred tank bioreactor stage (p < 0.05). Impeller of bent blade disc turbine had the highest comprehensive evaluation index
CFD during the fermentation process, with the optimal agitation speed and gas flow rate 400 rpm and 7.8 L/
Taguchi method min at low viscosity stage, and 400 rpm and 7.8 L/min at stages of medium and high viscosity.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction energy-consuming in a stirred tank bioreactor, which requires high

Aerobic fermentation is commonly employed in industries of
food, pesticide, medicine and cosmetic, etc. (Buffo et al., 2016). As
one of the most often used methods of process operation, stirring is
typically used in aerobic fermentation by enhancement of mixing
(Gill et al., 2008). A direct index for quality evaluation of aerobic
fermentation is product mass from the fermentation. But in order
to monitor the product mass, fermentation experiment has to be
carried out from beginning to end, which is time-consuming and
laborious. As an indirect index, gas holdup was frequently adopted
for quality evaluation of aerobic fermentation, especially the
fermentation in a stirred tank bioreactor (Riegler et al., 2019; Witz
et al., 2016). Higher gas holdup results in higher efficiency of mass
transfer, which contributes to increase of product mass
(Sardeshpande et al., 2017). Moreover, measure of gas holdup is
more convenient than measure of product mass. But stirring is
* Corresponding author.
** Corresponding author.
E-mail addresses: zhx1030@swu.edu.cn (X. Zou), liywu@swu.edu.cn (Y. Li).
power input to accelerate mixing for better heat and mass transfer
(Liu et al., 2019; Zheng et al., 2018a). Then for energy-saving pro-
duction of fermentation, excessive power input should be avoided
(Taghavi et al., 2011).
Many factors affect gas holdup and power consumption in aer-
obic fermentation. Among them, impeller type, gas flow rate,
agitation speed and viscosity of fermentation broth are often
studied (Choonia and Lele, 2013; Zou et al., 2012). Impeller type
directly affects the transfer process of oxygen and energy, and as a
result it affects synthesis process of microbial products (Tang et al.,
2015). Different impellers require different power consumption for
a certain aerobic fermentation. For a single-layer impeller stirring
system, radial flow impeller is presumed to have a good mixing
ability (Collignon et al., 2010). Rushton turbine (RT) (Ameur et al.,
2016), concaved blade disc turbine (CBDT) (Devi and Kumar,
2017) and bent blade disc turbine (BBDT) (Xia et al., 2009) are
representatives of the radial flow impeller. Impellers of CBDT, BBDT,
and modified CBDT or BBDT showed better performance of energy-
saving than impeller of RT (Luan et al., 2017; Zheng et al., 2018b).
Agitation speed directly affects gas holdup and power

https://doi.org/10.1016/j.jclepro.2019.05.083
0959-6526/© 2019 Elsevier Ltd. All rights reserved.
 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084 1075

consumption. High agitation speed promotes good gas-liquid Table 1

dispersion, and as a result it contributes to product mass increase Medium composition and proportion.

from aerobic fermentation, but it requires more power consump-
tion to stir the fermentation broth (Liu et al., 2016; Seo et al., 2014).
Gas flow rate represents amount of air that flows into the broth per
unit time, higher gas flow rate resulting more oxygen provided for
growth of microorganisms, and it significantly affects yield of
fermentation product (Brumano et al., 2017).
Pullulan fermentation of Aureobacidium pullulans belongs to
aerobic fermentation. The fermentation broth shows properties of
pseudoplastic fluid which is generally characterized by shear
thinning (Wucherpfennig et al., 2013). For fermentation process of
Aureobacidium pullulans, viscosity of fermentation broth increases
gradually due to the growth of microorganisms (Gabelle et al.,
2012). The published studies on aerated mixing of pseudoplastic
fluid were focused on pre-selected viscosity at a certain time of
fermentation (de Jesus et al., 2017; Sungkorn et al., 2012), and they
were lack of overall evaluation of the fermentation process. As a
matter of fact, fermentation broth of Aureobacidium pullulans un-
dergoes viscosity stages from low level to high one. Consequently,
overall evaluation and optimization of the fermentation process are
very important to practical use, considering the stages of viscosities
that the fermentation broth undergoes.
Computational Fluid Dynamics (CFD) is a numerical approach to
analyze and solve fluid involved problems. As for aerobic fermen-
tation in stirred tank bioreactors, CFD method can provide strong
capability to simulate flow of the broth, interaction of its in-
gredients (liquids and gases), and the resultant information of gas
holdup and power consumption can be obtained (Sungkorn et al.,
2012; Trad et al., 2017). Orthogonal-based Taguchi method and
statistical analysis make select of optimized combination and
quantitative evaluation of control factors possible. Combining CFD
and Taguchi method to solve the fluid involved problems was
widely applied in various fields, such as chemical engineering (Bao
et al., 2013), mining (Luo et al., 2016) and electric power (Wang
et al., 2018), etc. For a complex system, CFD combined with Artifi-
cial Intelligence (AI) methods, such as genetic programming and
automated neural network search provides an explicit solution to
process optimization (Garg et al., 2018b, 2018a), but lots of samples
are required for the combination (Shui et al., 2018).
The aim of this paper is to optimize aerobic fermentation pro-
cess in a stirred tank bioreactor so as to improve gas holdup and
power consumption, taking pullulan fermentation of Aureobasi-
dium pullulans as a case study. The novelty and contributions of this
paper include: (1) three representative viscosities and the corre-
sponding viscosity stages were innovatively defined for quantita-
tive evaluation of control factors on the fermentation process; (2)
the optimal parameters of impeller type, gas flow rate, and agita-
tion speed were obtained, which provided basic references to the
quantitative optimization of fermentation process.
2. Materials and methods
2.1. Microorganism and cultivation medium
Aureobasidium pullulans ATCC 201253 was used in this study and
it was obtained from College of Pharmaceutical Sciences, South-
west University. The compositions of the seed cultivation medium
and batch medium are presented in Table 1. The pH of the two
media were adjusted to 6.5 with 1 M HCl and the media were
autoclaved at 121
C for 30 min.
Four samples of 50 mL seed cultivation medium were inoculated
in 500 mL conical flasks for 48 h at 200 rpm on a rotary shaker
(HZQ-Q, Changzhou Aohua Instrument, China). Batch fermentation
was carried out in a 5 L stirred tank bioreactor (BIOTECH-5JG,
Medium Constituent Concentration (g/L)
Seed cultivation medium Sucrose 50.00
Peptone 3.00
K2HPO4 2.00
MgSO4 4.00
NaCl 2.50
FeSO4 0.01
Batch medium Sucrose 93.10
Peptone 3.00
NaCl 3.58
K2HPO4 3.00
FeSO4 0.02
BXBIO, China) with fermentation broth 3 L at 28 ± 0.1
C for 4 days.
The gas flow rate was maintained at 4.8 L/min. Agitation speed
increased from 400 to 700 rpm step by step, to guarantee enough
dissolved oxygen for an efficient fermentation.
2.2. Fermentation equipment
The fermentation equipment is composed of stirred tank
bioreactor, motor, control system, etc., as shown in Fig. 1. Di-
mensions of the bioreactor are: diameter of the tank T ¼ 175 mm;
height of the tank F ¼ 240 mm; height of fermentation broth
H ¼ 125 mm; width of baffles B ¼ 12 mm (4 baffles altogether);
height of baffles E ¼ 220 mm; clearance of baffles and tank wall
I ¼ 2 mm; diameter of agitation shaft d ¼ 12 mm; height of impeller
above tank bottom C ¼ 47.5 mm; height of air sparger above tank
bottom G ¼ 10 mm. The dimensions of impeller and air ring sparger
are shown in Fig. 2. Thirteen holes are evenly distributed along the
sparger.
2.3. Biomass concentration measurement
Biomass concentration was measured by means of Dry Cell
Weight method. Biomass suspension of an 8 mL broth sample in the
tube was centrifuged, rinsed, and dried to a constant weight. The
centrifugation was conducted at speed of 12000 rpm for 5 min,
using centrifuge of TGL-16M (Lu Xiangyi Centrifuge Instrument,
China). The biomass concentration of dry weight (g/L) was calcu-
lated as difference between weight of tube with and without dried
mass divided by the sample volume. The weighing was done with 3
replications by an electronic balance with precision of ±0.0001 g,
and mean weights were recorded.
2.4. Rheological measurement
Power-law model was used to describe rheological properties of
fermentation broth of Aureobasidium pullulans. Pseudoplastic fluid
was assumed for the broth, with Power-law equation expressed as
(Lebranchu et al., 2017; Wucherpfennig et al., 2013):
ma ¼ Kðga Þn1 (1)
where, ma is apparent viscosity, Pa$s; ga is apparent shear rate, s1;
K is consistency index, Pa$sn; n is flow behavior index.
The rheological parameters of the broth were measured first
from a shear rate of 0.1e300 s1, and then from a shear rate of 300
to 0.1 s1, using rheometer of DHR-1 (TA Instruments, USA). 3e5 mL
fermentation samples were taken and standard steel plate of
diameter 40 mm was selected for the measurement. 20 viscosity
data were measured at the pre-set shear rates. The ambient tem-
perature was set to 28
C, consistent with temperature in the
1076 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084

Fig. 1. Configuration of fermentation equipment: (a) physical apparatus; (b) schematic diagram. 1. Stirred tank bioreactor, 2. Ultrasonic distance sensor, 3. Motor, 4. Computer, 5.
Control system.

Fig. 2. Impeller and sparger: (a) 6-blade RT; (b) air sparger.

fermentation process. The data at the range of 0.1 to 1s1 was concentration; g/L.
discarded due to large error. Based on reliable viscosity data,
rheological parameters of K and n were obtained by fitting of
Power-law model. 2.6. Gas holdup measurement

2.5. Fitting of rheological parameters and biomass concentration
Three-parameter logistic model was adopted to fit rheological
parameters and biomass concentration of the fermentation broth,
and it is expressed as (Goudar et al., 1999):
Overall gas holdup was used to evaluate quality of the fermen-
tation in this study. The gas holdup was calculated from difference
between height of the broth level before and after the aeration in
the bioreactor, and it is expressed as (Ahmed et al., 2010; Prakash
et al., 2018):

K0 eaX Hg  H0
K¼
 (2) aG ¼  100 (4)
K0 Hg
1 1  eaX
Kf
where, aG is gas holdup, %; Hg is height level after aeration, mm; H0
1  nf is height level before aeration, mm.
n ¼ nf þ (3)
1 þ ðbXÞc The heights of the broth level were measured with 20 replica-
tions, using an ultrasonic distance sensor of NU200F18TR-S-1000
where, a, b and c are constants; Kf and nf are the final parameters with precision of ±0.01 mm (Jinci Technology, China), as shown in
of K and n; K0 is the initial parameter of K; X is biomass Fig. 1.
 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084 1077

2.7. CFD modeling 2.7.4. Numerical strategy

2.7.1. Governing equations Unstructured meshes were generated in ICEM CFD 18.1, and they
Euler multiphase model was used to investigate the two-phase were converted to polyhedral meshes in Fluent 18.1. Multiple
flow of fermentation broth. Here liquid was continuous phase, gas reference frame (MRF), as a steady-state method, was adopted to
was dispersed phase, and they were defined as phase l and phase g, solve the problems with rotating impellers (Varela et al., 2018).
respectively. Taking phase l as an example, its continuity equation Pressure-based solver with coupled algorithm was chosen for
and momentum equation are expressed as (Montante et al., 2008; improvement of rate of solution convergence. The solution was
Villiger et al., 2018): converged when residuals fell below 104, and at the same time,
velocity and gas holdup of a random selected point (45,80,45) had
v  
ða r Þ þ V, al rl !
no significant change (<1%) within iterations of 1000 steps.
vl ¼ 0 (5)
vt l l
2.7.5. Mesh independence verification
v !
ða r !
n Þ þ V$ða1 r1 ! n 1 Þ ¼ a1 Vp  V$t1 þ a1 r1 !
n 1! g þ F 1g
vt 1 1 1 It is necessary to investigate the independence of mesh. Three
(6) meshing schemes with different number of cells (scheme I: 232295
meshes; scheme II: 333641 meshes; scheme III: 465761 meshes)
where, ! vl is velocity, m/s; rl is density, kg/m3; al is volume frac- were accomplished for confirmation of mesh independence, and
tion; tl is stress-strain tensor, N/m2; p is pressure between the two velocities at the pre-selected point and torques acting on the
!
phases, Pa; Flg is interphase force term, N. impeller were investigated, as listed in Table 2. The results showed
!
Interphase force term Flg includes drag force, virtual mass that schemes II and III had better mesh independence than scheme
force, turbulent dispersion force, etc. In this study, only drag force I since the related velocities and torques from these two schemes
was considered because of the insignificant effect from other forces were quite close to each other. If cost of computation was taken into
(Wang et al., 2014), and Grace model was adopted to describe the consideration for solution of fluid flow of the fermentation, scheme
drag force (Liu et al., 2016). II was employed in the subsequent simulation.

2.7.6. Post-process of solutions

2.7.2. Turbulence equations Details of fluid flow parameters can be obtained from simula-
tion. The parameters include pressure, velocity, volume fractions of
The RNG kε model was used to describe the turbulence individual phases, torque acting on impeller, etc. Gas holdup can be
behavior of the fermentation broth. The k equation and ε equation calculated as value of gas volume divided by fluid volume, and
are expressed as (Yufeng et al., 2013): power consumption can be calculated from the torque acting on
! impeller as follows (Chen et al., 2016):
v v v vk
ðrkÞ þ ðrkmi Þ ¼ ak meff þ Gk þ Gb  rε  YM (7) pNMy
vt vxi vxj vxj P¼ (9)
30
! where, P is power consumption, W; My is torque, N$m; N is agita-
v v v vε ε
ðrεÞ þ ðrεmi Þ ¼ aε meff þ C1ε ðGk þ C3ε Gb Þ tion speed, rpm.
vt vxi vxj vxj k
ε2 2.7.7. Verification of CFD-based simulation
 C2ε r  Rε (8) The simulated results of gas holdup on selected conditions were
k
obtained from the pre-mentioned CFD method, and they were
where, xi and xj are positions in the ith and jth coordinate di- compared with the corresponding experimental results, as listed in
rections, respectively; Gk is generation of turbulence kinetic en- Table 3. As seen in Table 3, the gas holdup values of simulation and
ergy due to mean velocity gradients, m2$s3; Gb is generation of experiment were close to each other, and the mean value of the
turbulence kinetic energy due to buoyancy, m2$s3; YM is contri- relative error was 6.18%, which revealed that the CFD-based
bution of fluctuating dilatation in compressible turbulence to simulation was reliable for the fluid flow simulation of the
overall dissipation rate; Rε is additional term; ak and aε are the fermentation process. Still, the relative error needs to be focused
inverse effective Prandtl numbers for k and ε; meff is effective and considered. The reason for the influence on the error may be
viscosity; C1ε ; C2ε and C3ε are constants, and C1ε ¼ 1:42, C2ε ¼ due to the fact that the uniform size method was used and inter-
1:68 , C3ε ¼ 1:3. phase forces were idealized in the Euler multiphase model, which
made the simulation of size and dispersion of the bubbles not ac-
curate enough, and on the other hand, due to fluctuations in the
2.7.3. Initial and boundary conditions gas-liquid interface, the data of height obtained from the ultrasonic
distance sensor had a certain degree of fluctuation, which may also
Openings of the sparger and top free surface of the stirred tank cause error in the experiment.
were inlet and outlet of the fermentation model, and they were set
velocity inlet and pressure outlet as their boundary conditions
Table 2
respectively. Impellers and the agitating shaft were set as moving Results of mesh independence.
walls, other walls kept static by default, and no-slip boundary
Schemes Number of cells Velocity of the point (m/s) Torque of RT (N$m)
conditions were specified to all walls. Uniform size method was
adopted to set the bubble size. The average diameter of bubbles was I 232295 0.190 0.108
defined as 4 mm (Basavarajappa and Miskovic, 2016; Popa et al., II 333641 0.196 0.107
III 465761 0.195 0.106
2015).
1078 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084

Table 3
Comparison of simulated gas holdup with experimental results.

Selected conditions Gas holdup (%) Relative error (%)

Agitation speed (rpm) Gas flow rate (L/min) Simulated results Experimental results

400 4.2 3.14 3.41 7.92

400 5.4 3.94 3.75 5.07
400 6.6 4.72 4.24 11.32
400 4.8 3.54 3.43 3.21
500 4.8 3.84 3.91 1.79
600 4.8 5.21 5.65 7.79

2.8. Experimental arrangement

Orthogonal-based Taguchi method was applied to arrange ex-

periments. Taking impeller type, gas flow rate and agitation speed
as control factors, and gas holdup and power consumption as
evaluation index, experiments were designed according to an
orthogonal array L9(34). The experiments were conducted under
conditions of three representative viscosities.
Higher gas holdup and lower power consumption are preferred
for practical use. A comprehensive evaluation method was used for
the overall performance evaluation of fermentation process. The
comprehensive evaluation index is defined as:

0
 0

Ev ¼ h1 aG þ h2 1  P (10)

0
where, Ev is comprehensive evaluation index; aG and P0 are
normalized indexes of aG and P, respectively; h1 and h2 are
weighted values of aG and P, respectively. In this study, equal
weights were given to aG and P, namely, h1 ¼ h2 ¼ 0.5.
Fig. 4. Consistency index vs. fermentation time and their fitting curve, with R2 0.982
for Eq. (13). Three viscous regions of low viscosity, medium viscosity and high viscosity
were divided by dash lines.

3. Results and discussion

3.1. Analysis of rheological parameters and biomass concentration 0:026e0:267X

K¼
 (11)
1  0:034 1  e0:267X
The rheological parameters of fermentation broth with biomass
concentration and fermentation time were obtained from experi- 0:672
ments, as shown in Figs. 3 and 4. n ¼ 0:328 þ (12)
For consistency index and flow behavior index vs. biomass 1 þ ð2:881XÞ0:979
concentration, a 3-parameter logistic model was applied to exper- For consistency index vs. fermentation time, a 4-parameter lo-
imental data, and their regression equations were obtained: gistic model of growth curve was applied, and its regression
equation was obtained as well:

0:964
K¼  . 2:232 þ 0:985 (13)
1 þ Tf 60:206

where, Tf is fermentation time, h.

Fig. 3. Consistency index and flow behavior index vs. biomass concentration and their
fitting curves, with R2 0.966 and 0.975 for Eq. (11) and Eq. (12), respectively.
The resulted regression equations gave good fit to experimental
data of rheological parameters, with high coefficients of determi-
nation (R2 > 0.95). Consistency index characterizes viscosity of
fermentation broth (Kumar and Dubey, 2017; Osadolor et al., 2018).
Consistency index increases with growth of microorganisms in the
broth as fermentation time increases. In order to depict viscosity
change during fermentation and optimize the fermentation pro-
cess, 3 viscosity stages were defined by equally dividing consis-
tency index according to Eq. (13) and its fitting curve. The
representative viscosities at these stages were defined as low vis-
cosity, medium viscosity, and high viscosity, and their consistency
indexes were obtained by means of integral operation with
fermentation time at the corresponding viscosity stages, as shown
in Fig. 4. Then, the corresponding fermentation time and flow
 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084
behavior indexes of representative viscosities at different stages
were obtained from Eqs. 11e13. Finally, the rheological parameters
of fermentation broth at different fermentation time were obtained
as: low viscosity at 21.1 h: K ¼ 0.106, n ¼ 0.369; middle viscosity at
47.6 h: K ¼ 0.380, n ¼ 0.347, high viscosity at 76.5 h: K ¼ 0.629,
n ¼ 0.341. These representative viscosities were used as control
conditions in subsequent orthogonal experiments.
3.2. Single-factor experiments and analysis
Single-factor experiments were conducted to define factor
levels of gas flow rate QG and agitation speed N, and to verify the
CFD-based simulation.
The experimental data of gas holdup vs. gas flow rate and
agitation speed were obtained, as shown in Fig. 5. The ranges of QG
and N for the experiments were 3.0e7.8 L/min and 400e700 rpm,
respectively. The selections of the ranges referred to the recom-
mendations of fermentation equipment manual and cited literature
(Cunha et al., 2015). As can be seen from Fig. 5, gas holdup mono-
tonically increased with the increase of QG and N, and the increase
of QG contributed more to the increase of gas holdup than that of N
in the selected ranges. Then, ranges of QG 3.0e7.8 L/min and N
400e700 rpm were employed for the subsequent orthogonal
experiments.
3.3. Orthogonal experiments based on CFD modeling
3.3.1. Determination of control factors and levels
Based on single-factor experiments of gas flow rate and agita-
tion speed, the factor levels of QG and N were defined as: gas flow
rates 3.0 L/min, 5.4 L/min and 7.8 L/min; agitation speeds: 400 rpm,
550 rpm and 700 rpm. As for the control factor of impeller, impel-
lers of BBDT, CBDT and RT were selected, as shown in Fig. 6. The
dimensions of RT were detailed in Fig. 2. Diameter D of BBDT and
CBDT was the same with that of RT, and the other parameters were
in accordance with HG/T 3796. 5e2005 (Chinese Chemical Industry
Standard committee, 2005The factor levels were listed in Table 4,
and the experimental arrangement L9(34) of 9 runs was available
Fig. 5. Gas holdup vs. gas flow rate and agitation speed (experiment 1: fixed agitation
speed 400 rpm; experiment 2: fixed gas flow rate 4.8 L/min). Fermentation time was
73 h, rheological parameters were K ¼ 0.56, n ¼ 0.33; error bars showed standard de-
viation of data from 20 repetitions of experiments, above which different letters were
significant difference levels according to Duncan's Multiple Range Test (p < 0.05).
1079
for simulation, as listed in Table 5.
3.3.2. Distribution of gas holdup
Distributions of gas holdup from simulation at different vis-
cosity stages were shown in Fig. 7. As seen from Fig. 7, higher vis-
cosity resulted in higher gas holdup and more evenly gas-liquid
dispersion on the whole, partly because retention time of gas in-
creases with the increase of viscosity. Contours of the gas holdup at
different viscosity stages depicted change of gas-liquid dispersion
with viscosity and control factors. Two symmetrical high gas
holdup regions in the upper part of the impeller and near the baffle
were observed. Higher QG and N resulted in higher values of gas
holdup in these regions, which was of disadvantage to gas-liquid
dispersion for fermentation. Further attention should be paid to
improvement of local gas-liquid dispersion that was resulted from
better flow structure through optimal design of fermentation
system.
Swirling phenomenon existed in some runs, and it caused gas
above liquid surface to be swirled into the fermentation broth. The
phenomenon should be eliminated as far as possible. Baffles
benefited lessening of the phenomenon (Wo
jtowicz, 2014).
Nevertheless, the baffles in fermentation broth were incapable of
avoiding the swirling, primarily because of excessively high agita-
tion speed of impellers. Other reasons resulting in swirling may
include geometry of impeller, baffle size, and volume of fermen-
tation broth, etc. Further investigation should be focused on these
factors.
3.3.3. Effect of viscosity on gas holdup and power consumption
Results of gas holdup, power consumption, and evaluation index
at different fermentation time were obtained and plotted, as shown
in Table 5 and Fig. 8. The values of fermentation time map the
representative viscosities at different viscosity stages.
As shown in Fig. 8 (a), gas holdup increased with the increase of
viscosity to a large extent, with the 8th run being an exception. The
tendency was consistent with analysis of gas holdup distribution
reflecting retention time of gas in the fermentation broth. There-
fore, more attention should be paid to effect of viscosity on gas
holdup in the fermentation process. As shown in Fig. 8 (b), power
consumption slightly increased with the increase of viscosity,
which showed that the effect of viscosity on power consumption
was not significant within the range of viscosity studied. The rea-
sons for the small effect included small values of viscosity of the
fermentation broth of Aureobasidium pullulans and small volume of
fermentation broth. Furthermore, there were different perfor-
mances of gas holdup and power consumption for different runs at
the same fermentation time, and statistical analysis was required
for the performance evaluation. As shown in Fig. 8 (c), good
fermentation performance was largely achieved at medium and
high viscosity stages. The 8th run had the highest Ev at low viscosity
stage, and the 6th run had the highest Ev at medium and high
viscosity stages. Then, a comprehensive evaluation of Ev for these
runs at different stages was required.
3.3.4. Statistical analysis and process optimization
Range analysis and analysis of variance (ANOVA) were
employed to evaluate effects of control factors and their significant
levels. Based on the statistical analyses, the optimal combinations
of control factors at different viscosity stages were obtained as well.
The results of range analysis and ANOVA are summarized in Table 6
and Table 7, respectively.
At low viscosity stage, agitation speed had the highest level of
significance that affected evaluation index, and it was followed by
impeller type and gas flow rate in sequence. All control factors
acted as significant factors in the fermentation process (p < 0.05). At
1080 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084

Fig. 6. Geometric models of impellers.

Table 4 and they are normalized values of tL, tM and tH; tL, tM and tH are
Controls factors and levels. duration of fermentation time, and tL ¼ 36.5 h, tM ¼ 22.9 h and
Level Factors tH ¼ 36.6 h; kimL, kimM and kimH are values of k1 values of impeller
A B C
type in Table 5.
According to Eq. (14), the Evim values of RT, CBDT and BBDT were
Impeller type Gas flow rate (L/min) Agitation speed (rpm)
43.96, 52.59 and 53.94 respectively. Then, impeller of BBDT had the
1 RT 3.0 400 highest comprehensive evaluation index, and it was selected as the
2 CBDT 5.4 550
optimal impeller at all viscosity stages. Therefore, the optimal
3 BBDT 7.8 700
combinations of control factors were defined as: impeller of BBDT;
gas flow rate 5.4 L/min, and agitation speed 400 rpm at low vis-
cosity stage; gas flow rate 7.8 L/min and agitation speed 400 rpm at
medium and high viscosity stages, gas flow rate had the highest medium and high viscosity stages.
level of significance, and it was followed by agitation speed and Nevertheless, the analysis results and the corresponding
impeller type. The factor of gas flow rate was a significant factor at optimal combination rely on practical fermentation system and
high viscosity stage, while others were insignificant factors at weights of gas holdup and power consumption of the system. Then
medium and high viscosity stages (p < 0.05). a trade-off between gas holdup and power consumption should be
The optimal combinations of control factors were: A3B2C1 at low made for fermentation systems of other technical schemes. Besides,
viscosity stage, namely impeller of BBDT, gas flow rate 5.4 L/min, the CFD model in this study was valid for the fermentation systems
and agitation speed 400 rpm; A2B3C1 at medium and high viscosity within ranges of control factors focused on. For beyond inputs,
stages, namely impeller of CBDT, gas flow rate 7.8 L/min, and adjustments and modifications should be made. Furthermore, vis-
agitation speed 400 rpm. To achieve the optimization of fermen- cosity stages more than 3 and their corresponding data can benefit
tation process, gas flow rate and agitation speed can be adjusted to the process optimization, especially for optimization that is com-
the optimal combinations at different viscosity stages. Neverthe- bined with AI methods.
less, the impeller type was inconsistent at low viscosity stage with
that at other stages, and the impeller was unchangeable in the
whole fermentation process, the selection of impeller type should 4. Conclusions
be further considered.
For selection of impeller type, a comprehensive evaluation index In this study, by applying CFD-Taguchi method, effects of
was put forward, considering duration of fermentation time at impeller type, gas flow rate and agitation speed on pullulan
different viscosity stages, as expressed as: fermentation process by Aureobasidium pullulans were investi-
gated, and the optimal combinations of control factors were ob-
0 0 0
Evim ¼ t L kimL þ t M kimM þ t H kimH (14) tained. Main conclusions were as follows:

where, t0 L, t0 M and t0 H are weights of fermentation time at stages of (1) Three representative viscosities and their corresponding
low viscosity, medium viscosity and high viscosity, respectively, viscosity stages were innovatively defined as control

Table 5
Experimental arrangement based on L9(34) for simulation and results.

No. A B C Gas holdup (%) Power consumption (W) Evaluation index Ev/  100

L M H L M H L M H

1 1 1 1 1 1.38 2.02 2.51 4.29 4.74 4.93 46.97 45.75 45.41

2 1 2 2 2 3.11 4.30 4.91 10.25 11.02 11.33 44.87 44.48 44.92
3 1 3 3 3 5.84 7.76 8.64 19.55 20.33 20.64 41.92 43.08 45.92
4 2 1 2 3 2.84 3.39 3.76 10.18 10.54 10.72 42.59 39.03 38.07
5 2 2 3 1 6.80 8.68 9.18 19.80 19.94 20.18 50.00 51.16 51.34
6 2 3 1 2 2.56 4.42 6.13 3.84 3.93 3.98 59.21 66.11 75.28
7 3 1 3 2 4.76 5.76 6.09 16.15 16.74 16.98 42.25 38.64 37.40
8 3 2 1 3 3.09 3.09 4.35 3.29 3.29 3.34 65.84 58.06 63.81
9 3 3 2 1 3.49 5.06 6.01 7.73 8.07 8.19 56.04 58.79 62.25

Notes: L represents low viscosity stage; M represents medium viscosity stage; H represents high viscosity stage.
 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084 1081

Fig. 7. Distribution of gas holdup from simulation at different viscosity stages: (a) low viscosity; (b) medium viscosity; (c) high viscosity. The numbers on the left of each contour
represent runs in Table 5.
1082 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084

Fig. 8. Results of all runs in Table 5 at different fermentation time that represents corresponding viscosity stages: (a) gas holdup; (b) power consumption; (c) Comprehensive
evaluation index.

Table 6 Table 7
Range analysis of Ev at different viscosity stages. ANOVA of Ev different viscosity stages.

Stage Level A B C Error Stage Source f Sum of squares Mean sum of squares F Sig.

Low k1 44.59 43.94 57.34 51.00 Low A 2 155.46 77.73 20.59 0.046*
k2 50.60 53.57 47.83 48.78 B 2 165.66 82.83 21.94 0.044*
k3 54.71 52.39 44.72 50.11 C 2 259.16 129.58 34.32 0.028*
R 10.12 9.63 12.61 2.23 Error 2 7.55 3.78
Rank C>A>B Total 8
The optimal A3B2C1
Medium A 2 113.56 56.78 2.80 0.263
combination
B 2 345.15 172.57 8.52 0.105
Medium k1 44.43 41.14 56.64 51.90 C 2 247.12 123.56 6.10 0.141
k2 52.10 51.23 47.43 49.74 Error 2 40.52 20.26
k3 51.83 55.99 44.29 46.72 Total 8
R 7.67 14.85 12.35 5.17
High A 2 172.25 86.13 6.94 0.126
Rank B>C>A
B 2 666.57 333.29 26.84 0.036*
The optimal A2B3C1
C 2 459.81 229.90 18.52 0.051
combination
Error 2 24.83 12.42
High k1 45.42 40.29 61.50 53.00 Total 8
k2 54.90 53.35 48.41 52.53
k3 54.49 61.15 44.89 49.27
R 9.48 20.86 16.61 3.73
Rank B>C>A
conditions, by considering growth-curve of viscosity during
The optimal A2B3C1 the fermentation. Then quantitative evaluation of control
combination factors on the fermentation process was made.
(2) Biomass concentration of Aureobasidium pullulans was
correlated with consistency index K and flow behavior index
 L. Shu et al. / Journal of Cleaner Production 230 (2019) 1074e1084
n, and consistency index was correlated with duration of
fermentation time. The regression equations of correlation
gave good agreement with experimental results (R2 > 0.95).
(3) Effects of control factors and their significant levels on gas
holdup and power consumption were evaluated by means of
range analysis and ANOVA. All factors were significant at low
viscosity stage, and insignificant at stages of medium and
high viscosity except for gas flow rate at high viscosity stage
(p < 0.05).
(4) The optimal combinations of control factors were obtained
as: impeller of BBDT; gas flow rate 5.4 L/min, and agitation
speed 400 rpm at low viscosity stage; gas flow rate 7.8 L/min
and agitation speed 400 rpm at medium and high viscosity
stages.
(5) Based on results of the present study, better fermentation
process and control schemes can be developed to promote
the energy-saving production of Pullulan and a cleaner aer-
obic fermentation technology. Multi viscosity stages of Aur-
eobasidium pullulans fermentation and other microorganism
fermentation should be focused on in the future work.
Acknowledgments
The work was supported by the Natural Science Foundation of
China (Grant No. 31871783) and Fundamental Research Funds for
the Central Universities (Grant No. XDJK2018AC001).
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