Korean Society for Biotechnology and Bioengineering Journal 2022; 37(3): 107-111
Published online September 30, 2022 https://doi.org/10.7841/ksbbj.2022.37.3.107
Copyright © Korean Society for Biotechnology and Bioengineering.
Tae Wan Kim2†,, Sangmin Jung1†,, Chae Il Lim1, Si Jae Park3, Jinwon Lee1, Byung-Keun Oh1*, and Jeong-Geol Na1*
1Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 04107, Korea
2Department of Biotechnology and Bioengineering, Chonnam National University, Gwangju 61186, Korea
3Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul 03760, Korea
Here we developed new baffled flasks for stable and efficient gas-liquid mass transfer in small-scale aerobic microbial fermentation. For this purpose, various types of baffled flasks were designed, fabricated using 3D printer, and evaluated in terms of mass transfer performance and cell mass production. To investigate the mass transfer efficiency of gas according to the baffle type, three types of flasks with latticed-, curved- or swirled-baffles on the bottom were prepared. An experimental result of CO2 absorption in alkaline water in three types of baffled flasks indicated that latticed-baffled flask had the best mass transfer performance. The gas transfer rate was also observed to have a strong influence on the productivity of microbial cultures. When E. coli cells were cultivated for 6 h, the baffled flask with higher mass transfer rate yielded higher cell and lower acetate concentration. When correlated with the result of E. coli culture, the CO2 mass transfer performance, which is expressed in reciprocal of CO2 dissolution time, showed a linear relationship with the final cell concentration. In the newly done study, based on the cell concentration, the latticed-baffled flask with pentagon and hexagon was found to have 30.4% and 6.1% higher culture performance in terms of final cell mass than the conventional non-baffled and bottom-baffled flasks, respectively.
Keywords: shake flask, baffle design, aerobic microbial culture, 3D printing
Flask culture is one of the most widely used methods in bioprocess research [1-5]. Prior to the full-scale reactor-level study, flask cultures have been utilized mainly in small-scale experiments that require the screening for many variables. Such experiments include isolation of microbial strains, design of medium and optimization of culture conditions. There are various kinds of flasks depending on size, and shape of the baffle . Among them, baffled flasks, which are generally known to have high gas-liquid mass transfer characteristics by generating strong turbulence , have been widely used in most of aerobic cultures except for microorganisms sensitive to shear force.
Recently, gases such as methane (CH4), carbon monoxide (CO) and carbon dioxide (CO2) have attracted much attention as useful carbon substrates in microbial fermentations for the production of biochemical and biofuels [6-10]. Many researchers have conducted studies to screen microorganisms that can assimilate those gases and to establish the culture conditions. Since the solubility of CH4 and CO gases in aqueous solution is very low, the performance of the CH4- or CO-dependent gas fermentation is directly linked to gas-liquid mass transfer characteristics . Therefore, it is essential to develop efficient flasks with high gas-liquid mass transfer rate even in smallscale experiments. This is likely to assist in the development of strains for gas fermentation, investigating physiological characteristics of developed strains, and optimizing culture conditions.
In this study, with the consideration of turbulence generation, various types of baffled flasks were designed and fabricated using 3D printer. Afterwards, the experiment for CO2 absorption into the alkaline water was conducted in the prepared flasks to evaluate which types of flasks are suitable for efficient gas-liquid mass transfer. Finally, the effect of mass transfer of gas on microbial culture was investigated from the cultivation of
Ten kinds of baffled flasks with different shapes were designed and classified into following categories: 1) three conventional flasks with or without baffles, 2) two kinds of latticed-baffled flasks, 3) three kinds of curved-baffled flasks, and 4) two kinds of flasks with swirl type baffles (Fig. 1).
All flasks (600 mL volume) have same specification except the shape of baffles as shown in Fig. 2 and were designed by 3D modeling software (Solidworks 2017, Dassault systems).
The designed flasks were fabricated using 3D printer (LUGO M Printer, Formerspham Co., Korea), which includes a 0.40 mm single outlet and a heating bed. For 3-D printing, a transparent PLA (polylactic acid) filament (1.75 mm) was used as a flask material, and slicer program (ideaMaker, Raise3D, CA, USA) was used. After printing, the fabricated flasks were dried in an oven at 70°C for 15 min, and was later subjected to a test for liquid leak for 7 days. The results of the leak tests indicated that, no leakage occurred at all for 7 days in flasks fabricated under following printing conditions: layer height of 0.8 mm, 2 shell, first layer height of 0.25 mm, printing speed of 50 mm/s, a rectilinear patterned infill of 100% density, and a raft of 5 mm offset without support. Those leak-free flasks were used in CO2 absorption and microbial cultivation experiments.
In this study, titration experiments using CO2 gas were conducted to evaluate the gas-liquid mass transfer performance of the flasks . Fifty milliliters of the aqueous solution with a pH of 11 was added into the fabricated flasks and then 100 μL of phenolphthalein solution was fed into the flasks using a pipette. After confirming that the color of the solution in flask changed to purple, 20 ml of CO2 was injected into the flasks stirred at 150 rpm in an orbital shaker (SHK-30, Jung Biotech Co., Korea). The CO2 transfer rate was determined by measuring the time from the start of the injection until the pH of the solution in flask became 8.2 at which the color of solution became colorless.
Microbial flask culture experiments were carried out using
In this study, three types of baffled flasks were made of plastic material (PLA) using 3D printer (Fig. 1). The latticed-baffled flasks have the lattice-shaped baffles at the bottom. The curved-baffled flasks consist of wavy baffles at the bottom, allowing the fluid to flow smoothly after touching the baffle. The swirled-baffled flasks were fabricated to incorporate both latticed- and curved-baffle characteristics. Considering the possible effect on the experiment results due to the difference in base material, PLA flask of the same specification and shape as those of ready-made glass Erlenmeyer flask was fabricated and used as a control (Fig. 1(A)). The results from the CO2 titration experiment indicated that the performance of gas transfer depends on the types of baffle (Fig. 3). From the comparison of the time when the pH of the solution dropped from 11.0 to 8.2 by CO2 dissolution, it was clear that a longer time was needed for CO2 to be completely dissolved in flasks without baffles. On the other hand, the latticed-baffled flasks were observed to have the best CO2 transfer rate, while the curved-baffled flasks showed slightly lower gas transfer performance than the conventional bottom-baffled flasks. In the case of latticed-baffled flasks, despite of a small difference, the soccer-ball, which consisted of pentagon and hexagon, showed better gas transfer performance than the honeycomb, which consisted of hexagon only. The swirled-baffled flasks were observed to have moderate gas transfer performance between the curved- and latticed-baffled flasks. The difference seen in the results is explained by the degree of turbulence formation depending on the type of baffle.
Batch cultures of
Table 1 Cell growth and acetate production during 6h batch cultivation of
|Flasks||OD600||Acetate (g/L)||Specific growth rate (1/hr)||Acetate production rate (g/L/hr)|
|Unbaffled||4.19 ± 0.55||0.37 ± 0.04||0.59||0.06|
|4 bottom||5.27 ± 0.26||0.24 ± 0.01||0.62||0.04|
|6 vortex counter-clockwise||5.10 ± 0.08||0.24 ± 0.01||0.61||0.04|
|Soccer ball||5.73 ± 0.12||0.09 ± 0.02||0.63||0.02|
Fig. 4 shows the relationship between mass transfer performance and cell concentration. The reciprocal of CO2 dissolution time was shown to have a linear relationship with the cell concentration. Considering that kLa is the value of transferred gas amount divided by time, it was reconfirmed that the higher mass transfer coefficient, the greater the cell growth.
This study demonstrates the possibility of easily developing a tailor-made small-scale microbial culture system with decent gas-liquid mass transfer rate by applying 3D-printing technology. By the 3D printer, we could easily control the shape and number of baffles located at the bottom of the flasks, and develop the new baffled flasks with higher gas-liquid mass transfer rate than the conventional non-baffled and bottombaffled flasks. As a result, the highest cell growth was obtained in the new latticed-baffled flask. Since the continuous development of computational fluid dynamics and 3D-printing technology is expected, new flasks with desired properties such as better gas-liquid mass transfer performance could be tailored for different purposes.
In this study, we tried to develop a tailed-made flask culture system suitable for aerobic fermentation using 3D-printing technology. For this, various baffled flasks were fabricated from PLA material using a 3D printer, and the mass transfer rate of CO2 in each flask was evaluated. As a result, it was confirmed that the difference in CO2 mass transfer rate occurred due to the difference in the formation of turbulence depending on the number and shape of the baffles. In addition, in order to investigate the effect of transfer efficiency of gaseous substrate on the culture characteristics, aerobic batch culture of
This research was supported by C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the ministry of Science, ICT & Future Planning (NRF-2015 M3D3A1A01064926), the Sogang University Research Grant of 2022 (202219012.01), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1F1A1068541).
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