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.