We also conducted growth curve experiments in a bioreactor to investigate how cell growth varies under different reaction mixture volume scales. The bioreactor environment allowed us to maintain precise control over critical parameters such as temperature, pH, and oxygen levels, which are essential for optimizing cellular growth. As we scaled up the reaction mixture, we closely monitored the growth rates and overall biomass production to identify any significant differences compared to our lab-scale experiments. However, due to office hour constraints, we cannot monitor the growth rate during the 1st to 16th hour.

Experimental Set-ups

The bioreactor we used is Winpact One Fermentation System.

According to a study published in Frontiers in Bioengineering and Biotechnology (2023), the optimal conditions for Yarrowia lipolytica growth include a temperature of 30°C, an air flow rate of 1 LPM, and a dissolved oxygen concentration of 40%. We have maintained these conditions consistently to optimize growth.

The bioreactor condition:

• Initial pH: 6.4
• Bioreactor: 30˚C, 225rpm, 1 LMP air flow*, 40% Dissolved Oxygen
• Initial inoculum: 2 night Y. lipolytica in YPD, washed twice with PBS
  • 1mL cells resuspended in sterile water was used
• Growth media tested: YPD, with dextrose content is changed to 40g/L glycerol

Fig 2.1 Bioreactor Growth Curve

Figure 2.2 Comparison with conical flask scale

Growth in the bioreactor demonstrates a shorter time to reach the mid-exponential phase compared to lab-scale setups. However, we observed that the bioreactor resulted in a lower cell concentration, as indicated by Optical Density (OD), which we define as a poorer outcome. Given that the bioreactor is equipped with optimized environmental conditions and sufficient agitation, we anticipated that it would yield a higher final cell concentration.

Fig 1 The environmental conditions are monitored during the operation. The white curve is the pH value, indicating the fluctuations in pH and poor control

Fig 2. The photo record of the excessive foam production in the bioreactor

Several factors contribute to this unexpected outcome. The most significant issue is excessive foam formation, which can lead to the misreading of pH levels by the sensors. This foam not only obstructs accurate pH measurement but also hinders the appropriate addition of necessary acids and bases to maintain stable pH levels. Consequently, the pH readings may not reflect the true environment of the culture, resulting in inadequate pH control. Additionally, excessive foam interferes with the mixing process, causing inconsistent distribution of nutrients and oxygen, and contributes to increased shear stress on the cells. These factors collectively highlight the complexities involved in scaling up bioprocesses, emphasizing the need for careful management of foam and mixing conditions to optimize growth in bioreactor systems.

In conclusion, while our experiments indicated poorer results at the bioreactor scale compared to the lab scale, they still provided valuable insights into microbial growth dynamics. Notably, growth using glycerol consistently demonstrated a shorter time to reach the mid-exponential phase, highlighting its effectiveness as a carbon source. Additionally, glycerol achieved the highest maximum growth rates across our lab-scale trials, reinforcing its potential for optimizing growth conditions. Despite the challenges encountered during the scale-up process, these findings lay a solid foundation for further exploration into enhancing bioprocess performance. Our results suggest that glycerol is a superior choice for cultivating Yarrowia lipolytica, as it facilitates earlier attainment of the stationary phase, thereby promoting lipid accumulation.

• Repeat the experiments using different types and concentrations of carbon concentration to explore their effects on growth.
• Conduct growth experiments in larger-scale bioreactors to compare growth dynamics across different scales.
• Consider lowering the initial cell volume to extend and delay the exponential phase, allowing for better observation of growth patterns.
• Implement continuous dry mass determination to accurately monitor growth over time.
• Incorporate the monitoring of additional factors such as dissolved CO₂ concentration and dissolved oxygen levels to enhance overall growth conditions.

Frontiers in Bioengineering and Biotechnology. (2023). Enhancing the thermotolerance and erythritol production of Yarrowia lipolytica. Frontiers in Bioengineering and Biotechnology, 11, Article 1108653. https://doi.org/10.3389/fbioe.2023.1108653
Peterson, R. (1996). Lec notes: Population growth. University of Idaho. https://www.webpages.uidaho.edu/wlf448/peterson3.htm