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Evaluation of the Dissolved Oxygen Sensor Pill for Filamentous Organisms

Background

Streptomyces coelicolor, a well-studied model for filamentous organisms, exhibits pellet growth during fermentation. Pellet morphology directly influences oxygen transfer, nutrient availability, and overall fermentation efficiency. This study explores the impact of a novel Dissolved Oxygen (DO) Sensor Pill on the growth characteristics of S. coelicolor, including pellet size, oxygen transfer rates, and biomass dynamics, absorbed in cultivation using Pakula medium. Comparisons were also made to cultures with glass beads, a common practice to achieve dispersed growth in filamentous organisms.

The study provides valuable insights into the compatibility of the DO Sensor Pill with filamentous organisms and its potential utility in improving bioprocess control.

Results

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Fig 1. Dissolved Oxygen vs Respiratory Activity Monitoring System, RAMOS®

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Fig 2. DO vs. Biomass Measurement with Fixed Bins

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Fig 3. Microscopic images documented at defined time points: 23, 49, 71, and 137 hours.

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Fig 4. Scan showing direct comparison of cultivation with and without DO Sensor Pill taken at 137 h. While there are differences between the pellet sizes, the approach with the DO Sensor Pill still clearly shows pellets and no complete dispersion.

Materials & Methods

Cultivation Conditions:

  • Medium: Pakula Medium.
  • Parameters: Cultivation was carried out at 30°C, 250 rpm, with a 50 mm shaking diameter.
  • Flask Configurations:
    • Three SBI flasks and one RAMOS ® flask.
    • Offline shake flasks with and without the DO Sensor Pill for comparison.
    • Glass beads (~2.5 mm diameter) were used to promote smaller pellet formation for the comparison with DO sensor pill flasks.
    • shake flasks without baffles

Measurements:

  1. Biomass:
    • Measured at wavelengths of 622 nm, 645 nm, and 940 nm via the Multiparameter Sensor (MPS) fixed bins setting.
    • Samples were paused for collection and resumed after one minute.
  1. Image Analysis:
    • Pellet morphology was documented at defined time points: 23, 49, 71, and 137 hours.
    • Additional scans were performed on fermentation broth to observe pellet size and structure.

Conclusion

  1. Cultures with the DO Sensor Pill produced smaller, less densely packed pellets compared to the flask without DO Sensor Pill, reducing aggregation and improving oxygen transfer within the pellets. Similarly, the use of glass beads achieved dispersed growth with uniformly smaller pellets than the pellets produced in the culture with the DO Sensor Pill.
  2. Biomass increased initially as pellets formed but declined following glucose depletion, with DO data clearly indicating a correlation between glucose depletion and oxygen transfer dynamics. The reduced pellet density facilitated better oxygen penetration.
  3. Microscopic imaging detected significant visual differences in pellet size and density, with larger pellets observed in cultures without the DO Sensor Pill.
  4. Cross-species comparisons conclude that the DO Sensor Pill did not cause mechanical stress in other filamentous organisms, such as Ustilago maydis, as evidenced by similar pellet morphology observed both with and without the DO Sensor Pill.

Summary

The DO Sensor Pill demonstrated significant advantages in improving oxygen transfer and mitigating oxygen limitations in filamentous organism cultivation. By reducing pellet size and promoting more dispersed growth, it ensures better oxygen penetration, which is critical for enhanced metabolic activity. These results underline the potential of the DO Sensor Pill as a valuable tool in bioprocess optimization, specifically for filamentous organisms like Streptomyces coelicolor.

This research was conducted by:

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“Incorporating SBI’s pH and DO flow cells into our system removed the need for manual sampling, saving us time, reducing the risk of contamination, and providing information on how the cells are growing even when we are not in the lab. With availability of this more detailed view of our culture, we can make informed improvements to our cell expansion process.”

-Kitana Manivone Kaiphanliam (Washington State University)
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