The escaping of genetically modified organisms from labs is a serious problem since it will bring up unpredictable impacts to our ecosystem. We take biosafety very seriously when we design every parts of our experiment and the design of our production line. Our design of our production line insure that there's no genetically modified yeasts (also non-genetically modified yeasts) could survived and pass the whole production line and leak into the environment or residue on our products. We also dealt cautiously with wastes produced from the experiment and participated in safety training, to minimum the chance of causing biohazard pollution, or simply hurt ourselves.

Filtering system

  The average size of the P. pastoris is about 4–6 μm (Gmeiner, C. et al. 2015), the filter we use is 33 kDa, which is equivalent to the size of tens of nanometers (Bacher, G. ey al. 2001), enough to block all the yeasts form leaking out the fermentation tank.

Heating system

  According to the literature (Martínez, D. et al. 2015), heating at 70 °C for 1 day or incubate at 30 °C for 48 hr, is sufficient to kill all the yeasts. The minimum operating temperature of Extrusion Granulation will be over 110 °C. Therefore, no yeast could survived in the production line.

Following are our heating tests:

Figure1: P. pastoris yeast heated at 30 °C for 1 day , and incubated at 30 °C for 48 hr.
(left: 0.005, right: 0.05)

Figure2: P. pastoris yeast heated at 50 °C for 1 day , and incubated at 30 °C for 48 hr.

Figure3: P. pastoris yeast heated at 70 °C for 1 day , and incubate at 30 °C for 48 hr.

  We found that our P. pastoris not survival after were heated at 50 °C for 30 minutes. Thus, our production line heat process would kill P. pastoris passing through the filter.

Reference

Gmeiner, C., Saadati, A., Maresch, D., Krasteva, S., Frank, M., Altmann, F., … Spadiut, O. (2015). Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase. Microbial Cell Factories, 14(1). doi:10.1186/s12934-014-0183-3

Bacher, G., Szymanski, W. W., Kaufman, S. L., Zöllner, P., Blaas, D., & Allmaier, G. (2001). Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses. Journal of Mass Spectrometry, 36(9), 1038–1052.doi:10.1002/jms.208

3. Martínez, D., Menéndez, C., Echemendia, F. M., Hernández, L., Sobrino, A., & Trujillo, L. E. (2015). Kinetics of sucrose hydrolysis by immobilized recombinant Pichia pastoris cells in a batch reactors. J Microb Biochem Technol, 7, 294-6.