INTRODUCTION
Over the decades, microorganisms have been used as “mini-factories” in
biomanufacturing to aid in the diversity of metabolic pathways and their
accompanying ability to transform a wide range of renewable raw
materials into value-added compounds via fermentation [1-3]. In the
quest to obtain DHA from fish oil, species like Schizochytriumsp. have been widely used in liquid fermentation due to their
short growth cycle and high cell density cultivation in bioreactors
[4]. To optimize the culture, the screening of strains likeSchizochytrium sp. has been done from shake flasks to 5L
fermenters. However, this process has many issues, including low
optimization throughput, high labor intensity, and unsatisfactory
optimization results, which are related to the structure of shake flask
and the regulation of parameters in the fermentation process [5-6].
Microplates are commonly used to establish high-throughput technologies
in cell line and process development due to their high throughput, low
labor intensity, and ease of automation [7-9]. However, the existing
microtiter plate can only reach a KLa of 0.32
s-1, making it challenging to achieve high-quality
fermentation and cultivation [10-11]. Therefore, improving
the microtiter plate to adapt to the high-throughput screening of
DHA-producing strains is not only an academic goal, but also an urgent
need for industrial practice.
The liquid volume in microplates is usually very small, and in most
systems, it is impossible to control key system parameters such as
KLa and turbulence flow energy [10,12].
Additionally, the lack of online monitoring during fermentation makes
such systems difficult to use and understand [13]. However, with the
rapid development of CFD, its application in biological fermentation has
received more attention. By using mathematical modeling and numerical
solutions through a computer, it is possible to quantitatively
characterize the flow field properties in a bioreactor [14-15].
Therefore, CFD technology has been widely used to simulate flow fields
in bioreactors to obtain engineering parameters such as mass transfer,
mixing, and shearing [16-18]. Many studies have used CFD to redesign
various types of bioreactors through structural improvements and
hydrodynamic optimization to meet the needs of microbial culture in
industrial applications [19-21]. Therefore, utilizing CFD to
characterize key performance indicators like the oxygen mass transfer
coefficient, specific surface area and volume power input can visualize
the entire growth process of microorganisms and regulate the parameters
to optimize the conditions for achieving the best culture effect.
The cross-sections of existing microtiter plate are typically square or
circular, which cannot provide sufficient oxygen supply, a critical
parameter for microbial screening and process development [22-23].
In addition to increasing the vibration diameter and frequency and
reducing the volume of loaded liquid,modifying the geometry of the
bioreactor is also an effective way to achieve high efficiency of oxygen
transfer and mixing [24]. Many studies have shown that changing the
shape of the microtiter plate or introducing baffles can significantly
increase the maximum oxygen transfer capacity. For example, Delgado et
al. found that a bioreactor with baffles, could increase the maximum
oxygen transfer capacity by a factor of 5 to 10, even at lower vibration
frequencies [25-28]. However, it has been reported that the
parameters of the microbial growth process have not been well
characterized, and the reproducibility of growth is poor [29].
Moreover, excessive increases in the vibration frequency during
fermentation culture to enhance oxygen supply and mixing can lead to
liquid splashing, gas transport restrictions and fermentation pollution
[30]. Therefore, in many cases, vibratory bioreactors with other
geometries or baffles are not widely used.
This research established a high-throughput screening system for
producing DHA based on CFD technology. It involved modeling and
analyzing the oxygen supply level and mixing effect of microtiter plates
with different geometries, to evaluate key parameters such as oxygen
transfer coefficient, turbulence dissipation rate and volume power
input. In addition, the splash height and overflow level of the liquid
around the rocking orbit during vibration culture were also monitored in
real-time to ensure conditions were stable. The operation parameters
were then optimized using the response surface method, and a microplate
was constructed using 3D printing technology for culture experiments.
The superiority of the hexagonal microtiter plate with six baffles was
determined. This research was able to achieve high-throughput screening
for DHA-producing strains, which has significant implications for
scaling up DHA production in the industry.