Team:USP-Brazil/Results:FreezeDry

Results

Lyophilization

Defining the best cryoprotectants

We didn’t found on literature protocols or references about lyophilization of Pichia pastoris, but—as the baker’s dry yeasts may confirm—the protocols for yeasts are abundant. Using as reference the known methodologies for Saccharomyces cerevisiae and other yeasts [4], we tested combinations of two cryoprotectants for Pichia’s lyophilization: powered milk and monosodic glutamate (see our Notebook).

Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the lyophilizator), the following results were obtained:

Figure 1:We serially diluted the resuspended lyophilized samples: each section of plate corresponds to an ordered area of (three)10 microL drops from solutions diluted in different orders (as could be seen in the Milk + Glutamate plate, the order of dilutions is from right to left and up to dowm).

Table 2: CFU for each test in 1.5 mL tubes. 24h lyophilization process with 1 mL of cell suspension.

Although threalose was not used—what is also a good protectant for yeast species [4]—, the result showed itself interesting enough for a cheaper way to make lyophilized Pichia. Since lactose is not metabolized by this yeast [5], this might not affect P_AOX1 activation if the powdered milk does not have residual glucose quantities. milk doesn’t have residual glucose quantities. As could be seen on the graph next subsection, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying.

Ethanol Resistance after Lyophilization

To test the usefulness of this preservation method for the application using ethanol solution (alcoholic drinks), we tested the survival of P.pastoris cultures in solutions with different ethanol concentrations. The ability to survive to ethanol medium even after a stressful lyophilization process is a determinant characteristic that our chassis must have. Using the same methodology to count UFCs as before (like the previous image), and concentrating two times the 1 mL cultures using a table-top centrifuge before the lyophilization procedure—in order to have a larger survival population—we achieved very interesting results, with surprisingly a high survival percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!