Team:USP-Brazil/Results:FreezeDry

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 lyophilization machine), the following results were obtained:

YPD	Glutamate
Milk	Milk + Glutamate

Figure 1:Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).

Although threalose was not used— witch is also a good protectant for yeast species [4]—, the results showed that milk + glutamate is 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. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, 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 process.

Ethanol Resistance after Lyophilization

To test this preservation method for our intended application, where the yeast will be resuspended 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. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL cultures to 50% of its volume before the lyophilization process—trying to get larger survival rates— showed very interesting results.

After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four hours before plating in YPD, which was not much different from the 10% ethanol resuspension result after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).

Figure 2: cellular density variation on lyophilization process

When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water, we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate. In our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours, after the use of the device (Figure 3).

Figure 3: relative ethanol resistance 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.

H2O t=0h	H2O t=4h
Ethanol 10% t=4h	Ethanol 12.5% t=4h

Figure 4: Mind the difference between the dilutions orders contained on each plate—notations at the plates centers.

Storage time

The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, to efficiently address the project’s challenge, Pichia pastoris must be capable to do the same. We resuspended some samples from the first lyophilization of “ethanol survival test” after a couple of weeks. We again surprisingly achieved very good results, showing no significant variation of cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test (see graph below).

Figure 5: Relative percentages to the same control of the first ethanol lyophilization test.

A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.

We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.