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:

YPD	Glutamate
Milk	Milk + Glutamate

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— witch 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 metabolised 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)!

Figure 2: celular density variation on lyophilization proccess

Figure 3: relative ethanol resistence after lyophilization. Using the “Control” (see graph before) as reference.

We used a non-lyophilized serial diluted (YPD) plate of Pichia culture as a control. The plate of “t = 0h” was the immediately resuspension of free-dried P. pastoris on YPD plate and the “t = 4h” plates were resuspensions 4 hours after the “t = 0” resuspension. This was done to simulate the possible scenario with our detector—when after some couple hours the output might be come out. As expected, the survival of Pichia drops critically in a solution of higher concentration of ethanol than 10% [6]. This also corroborates with the Pichia’s grow curves and ethanol test plates showed previously. Another test was done, in same conditions, with another ethanol concentration more closely to 10%, and the result was maintained as image below shows.