RESULTS AND DISCUSSION
3.1 Flow field prediction and CFD model
validation
In order to test the reliability of CFD simulation and calculation
algorithms, preliminary simulations were carried out, and the model was
verified by comparing the
KLa and \(P/V\) results of the simulated gas-liquid
interface with the results calculated by empirical formulas in the
literature. The grid cells of the microtiter plates were divided into
approximately 550,000 cells, with verified independence(Fig.S1 ). The results of the simulation were shown inFigure 2 . It can be clearly seen in the Figure 2A and2B that the CFD simulation analysis results were very
consistent with the results in the experimental literature. The
predicted values differed from the results of the experiment, due to the
slight differences in the fluid characteristics of the fermentation
broth in experimental culture and the CFD. The liquid phase in the CFD
simulation used the rheological properties of pure water, with a density
(ρ) of 998.2 kg/m3, a viscosity (θ) of 0.01
m2/s, and a liquid surface tension (σ) of 0.0728 N/m.
There was good consistency between the simulated value and the
experimental value (p<0.05), indicating that the CFD model could
realistically reflect the data in the experiment.
In addition, the flow field prediction diagram of microtiter plate
sloshing was displayed by CFD to characterize the actual flow field at
the gas-liquid interface. The specific movement of the liquid in the
microplate was shown in Figure 2 , with blue parts representing
the liquid phase and red parts representing the gas phase.Figure 2C displayed the
contours plot on the
cross-section, and Figure 2D showed a three-dimensional motion
model of the microtiter plate. By tracking its movement during orbital
vibration, both figures clearly illustrated the real situation of fluid
movement in the experiment. During the rotational movement, due to the
action of centrifugal force, the liquid moved towards the wall, and the
gas-liquid interface area increased rapidly under strong vibration
conditions. After the orbital cycle was complete, the liquid in the
microplate circulated and reciprocated. Throughout the study, it was
assumed that the gas-liquid interface was located on an iso-surface with
a liquid phase volume fraction of 0.5. At the same time, the images of
the movement track also verified the authenticity and reliability of the
CFD model.
3.2 Performance evaluation of conventional microtiter
plates
Most of the microtiter plates used in current studies have circular or
square cross-sections. It has been proven that the oxygen transport
efficiency of square cross-sectional microtiter plates is significantly
higher than that of circular microtiter plates [40]. Therefore, only
square cross-sectional microtiter plates were used in this section to
explore and evaluate their performance during the culture and
fermentation of strains. During experiments, the growth of
microorganisms led to rheological changes in the fermentation broth. The
changes in density, viscosity and surface tension must be considered
during culture, in which the surface tension has a significant effect on
the mass transfer efficiency between gas and liquid [41]. As shown
in Table 1 , the surface tension of the fermentation broth
increased from 0.076 N/m to 0.103 N/m during the entire fermentation
process. This rise had a significant impact on mass transfer and mixing
performance between gas and liquid. Therefore, the phenomenon must be
discussed in depth to explore the applicability of microtiter plates.
In this study, the changes in rheological properties of the fermentation
broth of strains were simulated and analyzed in ANSYS Fluent. The
simulated environment was performed at 300 rpm and 15% liquid load, and
the results were shown in Figure 3 . With the passage of culture
time, the oxygen supply efficiency and mixing performance of the
microtiter plate gradually decreased. The average turbulent dissipation
rate (ε) also decreased from 0.45
m2/s3 at 0 h to 0.24
m2/s3 at 24 h, which greatly limited
the growth of the microbes. The strain cultured in conventional
microtiter plates was sampled after 24 hours, and it was observed
microscopically that the biomass did not show a well-growing trend. The
diameter of the cell was small, and the distribution was sparse. This
growth condition resulted in low biomass and poor microbial activity,
preventing the strains from meeting the needs of high-density
fermentation, which would also have a negative impact on DHA production.
Therefore, it is of great significance to optimize the existing
microtiter plates to adapt to the culturing and screening of DHA
producing strains.
3.3 New high-throughput bioreactor
design
3.3.1 Reactor geometry
design
Previous studies have demonstrated that the cross-sectional geometry of
microtiter plates significantly affects their mass transfer and mixing
properties [42]. In this section, the CFD technology was used to
model microplates with different geometries and analyze them
numerically, which explained the function and importance of geometry in
microbial culture. Figure 4A presented a series of microtiter
plate models with increasing edges, having a cross-sectional area
limited to approximately 60 mm2 and a total volume of
1.2 mL per titer. A vibration frequency of 150 rpm and a liquid charge
of 15% were used for the numerical solution to ensure the consistency
of reaction parameters. The influence of different reactor geometries
was analyzed. Real-time monitoring of the microtiter plate’s oxygen
transport efficiency was performed using Equations 3 and 4 in Fluent,
and its mixing performance was monitored in real-time using Equation 6.
The results were output after 5 seconds of motion at a vibration radius
of 3 mm, as shown in Figure 4 .
The results of the calculation for mass transfer related parameters
KL, a, and KLa can be seen inFigure 4B . As the cross-sectional geometry of the microtiter
plate changed from quadrilateral to hexagonal and gradually increased to
circular, the data for oxygen transport efficiency fluctuated, with the
relevant parameters reaching their highest values in the hexagonal
microtiter plate. When the number of edges was increased from the
conventional quadrangle to a regular hexagon, KLa rose
significantly. Since KL and a in the miniature vibrating
bioreactor contributed almost equally to KLa, the three
parameters performed consistently. These parameters reflected the best
performance in hexagonal microplates. In subsequent geometry
modification, the mass transfer efficiency gradually decreased and
finally reached a minimum oxygen transport efficiency in the round
microtiter plate, which was also consistent with other studies. The
oxygen transfer efficiency of the square cross-sectional microtiter
plate was significantly higher than that of the round microtiter plate,
and the mass transfer related parameters of the round microplate were
far inferior to those of other geometric reactors. In addition, the
turbulent flow kinetic energy (k), turbulent energy dissipation rate and
volumetric power consumption of the mixing performance were also
characterized in Figure 4C . Similar to the changes in mass
transfer parameters, as the number of sides of the reactor gradually
increased from quadrilateral to hexagonal, the mixing performance was
gradually optimized. Subsequently, in the regular heptagonal shape, the
mixing performance continued to improve slightly, because more edges and
corners affected the intensity of the internal movement. However, this
would lead to the splash of fermentation broth in the experiment,
increasing the risk of contamination. In simulations that continued to
increase the number of microplate edges, this short-lived improvement in
mixing performance disappeared and was replaced by a similar trend to
the mass transfer parameters, which reached a minimum in round
microplates. In conclusion, hexagonal microtiter plates showed superior
mass transfer and mixing performance in the simulation, which laid the
groundwork for future development of screening and culturing of DHA
producing strains.
Moreover, the contour diagram of the gas-liquid interface of microplates
in motion was characterized in CFD-Post to demonstrate the superiority
of hexagonal microtiter plates in mass transfer and mixing performance
(Fig.S2 ). The figure showed the differences in the speed of
liquid movement in different geometries of microplates. In hexagonal
microplates, the liquid had the fastest rate of movement. Additionally,
the liquid movement trend in several microplates showed consistency. The
movement speed near the bottom of the microtiter was low, while it was
relatively high at the upper end. The specific movement of the liquid
was caused by the applied centrifugal force. The superiority of the new
bioreactor was also verified in the velocity contours of the liquid,
which were consistent with the previous simulations. However, modifying
the geometric shape of the microtiter plate alone is far from sufficient
to meet the conditions of culturing and screening the DHA producing
strains, and further improvements will be necessary in subsequent work.
3.3.2 Structure and reaction parameter
optimization
In order to obtain optimal growth conditions, multiple parameters need
to be optimized for the growth of DHA producing strains. However,
gradually optimizing reaction parameters in software or experiments is a
complex and difficult task. The mathematical and statistical response
surface methodology (RSM) technique is a widely used method that
consists of a set of mathematical techniques to describe the
relationship between input and output parameters for modeling and
optimization purposes. This RSM model estimates the combination of the
input parameters yielding an optimal response through fast-running
approximation of the simulation process [43]. In this study, the
rotational speed, liquid filling volume and the number of baffles were
regarded as three independent variables A, B and C, KLa
and P/V were used as the result variables, and the RSM model was
established based on the numerical calculation results of CFD. A
complete experimental design matrix was given, and CFD-based simulation
numerical experiments were carried out according to the experimental
plan. The final results showed the effectiveness of each parameter and
the influence of its interaction on the mass transfer and mixing
performance. In subsequent work, ANOVA was performed to assess the
quality of the developed model, as shown in Table 2 . Based on a
95% confidence level, the results of the second-order quadratic models
of KLa and P/V have significant F and P values
(<0.05), which showed that the model constructed in this study
was suitable for the range of variables studied. In the results of ANOVA
in Table 2, the coefficients of determination R2 were
0.9803 and 0.9775, indicating that the model had a perfect correlation
between the independent variables and the response. The corrected
R2 were 0.9550 and 0.9486, indicating that only about
5% of the change could not be explained by the model.
The three-dimensional response surface was plotted based on the fitting
second-order polynomial (Fig.S3 ). The influence of the baffle
number, liquid charge and rotation speed on the changes of
KLa and P/V was clear. Consequently, the interaction
between the vibration velocity and the number of baffles significantly
affects the mass transfer and mixing performance of the new bioreactor.
The number of baffles and shaking frequency of the bioreactor were
positively correlated with mass transfer and mixing. With the increase
of the baffles and rotation speed, the performance of the bioreactor was
gradually optimized. On the contrary, the liquid filling volume was
negatively correlated with the mass transfer and mixing performance, and
the performance of the bioreactor tended to decrease with the increase
of liquid load. The optimal conditions recommended by the CFD-based RSM
model included 6 baffles, 15% liquid load, 800 rpm, maximum
KLa level of 0.61 s-1, and P/V of 2364
W/m3, which were sufficient to meet the growth
conditions of DHA producing strains. The splashing phenomenon that might
occur during the culture was also explained in subsequent model
verification experiments. In summary, this study completed a method
combined with the experimental design of the response surface on the
framework of CFD technology to obtain the optimal culture conditions for
DHA producing strains in a novel high-throughput bioreactor.
In this section, the superiority of the new high-throughput bioreactor
for culturing and screening of DHA producing strains was established on
the basis of CFD-RSM technology. Furthermore, simulation verification of
CFD and experiments with solid microplates were conducted. The optimal
reaction parameters given by the RSM model were numerically solved and
compared for conventional quadrilateral microplates, hexagonal
microplates, and the novel bioreactor using Fluent. The mass transfer
and mixing levels of the new bioreactors were significantly higher than
those of the other two microtiter plates. To further observe the
superior performance of the new bioreactor, the liquid phase motion
speed of the three microplates was derived in CFD-post. And planes of
different heights were selected to reveal the distribution state of the
liquid phase (Fig.5 ). Figure 5A showed that the motion
state of liquid in the new bioreactor is more intense and uniform than
that of the other two reactors, and the movement speed at parallel time
is also significantly higher than that of the other two, which is
conducive to the oxygen transport and nutrient absorption during the
strain growth. In Figure 5B , several planes were selected
inside the bioreactor. During the vibration movement, it was found that
the liquid level height was higher in the new reactor. The closer it was
to the ostiole, the better liquid phase distribution, compared with the
other two reactors. The liquid phase distribution was more uniform in
the new reactor rather than being limited to the wall surface, which
attributed to the different geometric structure. All in all, the new
bioreactor showed higher mixing and mass transfer levels than
conventional reactors during simulations, and this improvement was
sufficient to meet the culturing and screening needs of DHA producing
strains.
3.4 Utilization of the novel microplate for screening DHA
producing
strains
Based on 3D modeling and printing technology, this study established and
printed a new microtiter plate in equal scale. Three DHA producing
strains stored in the laboratory were screened using the new microplate.
The culture medium described above was used to cultivate the three
strains in both conventional and novel microplates for 48 hours. The
biomass of the fermentation broth was measured at 0, 12, 24, 36, and 48
hours. The results were shown in Figure 6A . The three species
showed different growth states and trends over the culture time in the
microplates. Under the same culture conditions, the three DHA-producing
strains showed no significant difference in conventional microplates,
and their biomass was lower than that of the novel microplates at the
end of the culture. However, in the new microplates, the growth state
and rate of Schizochytrium sp were significantly higher than
those of the other two strains, and the biomass of Schizochytriumsp was 20% higher than that of Aurantiochytrium sp. andThraustochytrium sp. at the end of culture. This phenomenon
indicated that the oxygen level and mixing capacity provided by the new
microtiter plate were better than those provided by the conventional
microtiter plate in the process of constant temperature oscillation
culture. There was no liquid spatter phenomenon during the entire
culture process, which facilitated the growth of microorganisms. In
addition, the new bioreactor had a larger cell diameter and higher cell
density, while the conventional reactor had a small cell morphology and
sparse cell density, due to oxygen dissolved limitation and uneven
mixing during the culture process. In conclusion, the performance of the
new bioreactor is significantly superior to that of the conventional
bioreactor in both simulation experiments and laboratory experiments. In
the process of vibration culture, it provides sufficient oxygen level
and high mixing efficiency for the growth of strains, which is conducive
to the high-throughput culturing and screening of high-yield DHA
strains.
3.5 Optimization of culture conditions by novel
microplates
On the basis of screening out a high-quality strain, the new microplate
was used to optimize the culture medium in order to increase the DHA
yield of fermentation. Design-Expert software was used to optimize the
content of monosodium glutamate (MSG), yeast extract (Yeast) and glucose
(Glu). Nile red staining reagent was used to quantitively characterize
the biomass, and the fluorescence intensity value was measured and used
as the regulatory variable. The RSM model provided 17 kinds of
experimental designs. Three groups of parallel experiments were
conducted according to the recommended experimental design. After 48 h
of culture, the samples of three groups were stained for measurement of
fluorescence intensity (Table 3 ). Consequently, the optimal
medium conditions included 20g/L MSG, 20g/L Yeast and 60g/L Glu. In
order to verify the accuracy of the experimental design recommendation,
at the end of the fermentation, the growth state of the strain was
observed and recorded under a fluorescence microscope, as shown inFigure 6B . In the following work, Image J was used to process
the pictures and calculate the average fluorescence density and the
average diameter of the microorganisms under each different regulation
medium state. The average fluorescence density represented the biomass
of the strains, and the average diameter quantified the growth state of
the strains. Considering the comprehensive comparison between the two
parameters, the actual growth state of the strain under the regulation
of the medium was observed, and the numerical results were shown inTable 3 . The fluorescence images of the strains were consistent
with the predictions of the RSM model. Under the recommended
experimental design, the density and diameter of the strains were
generally larger than those of other experimental designs. This study
efficiently optimized the growth parameters of DHA-producing strain
culture by using a newly designed microplate combined with response
surface design, which is of great significance for guiding industrial
fermentation for DHA in the future.