Cryopreservation of Pyramimonas sp. (KOPRI AnM0046) which has Ice Crystal Controlling Activity

Abstract

Korea Polar Research Institute (KOPRI) has polar microalgae culture collection. Polar microalgae thrive under extremes of temperature, salinity, and light. One factor that they survive is concerned with ice-active substances (IAS). We have been investigating IAS of KOPRI Culture Collection of Polar Mciroalgae (KCCPM). However, there are some problems because the culture collection is maintained by subculturing. Continuous serial cultures cause genetic drift and many chances for contamination. Pyramimonas sp. (KOPRI AnM0046) of KCCPM was obtained from Maxwell Bay, King George Island, Antarctica. In order to measure IAS ability, we carried out IAS assay. As a result of ice crystal controlling activity test, we found out KOPRI AnM0046 has ice crystal controlling activity. To preserve unique IAS character of polar microalgae KOPRI AnM0046 without genetic drift, we also carried out cryopreservation of KOPRI AnM0046. Cryopreservation takes place both plasmolysis and inner ice during freezing. It is concerned with cooling rate. In order to avoid these problems, optimization of cryoprotectants (CPAs) concentration and cooling rate is important. To determine the toxicity of various CPAs to KOPRI AnM0046, the following two variables were evaluated: five CPAs (dimethyl sulphoxide, DMSO

ethylene glycol, EG

glycerol, Gly

methanol, MeOH

and propylene glycol, PG), and four concentrations (10, 20, 30, 40%

v/v). As a result, the toxicity of CPAs was observed over 30%. Cryopreservation of KOPRI AnM0046 was acted by (a) uncontrolled direct freezing in liquid nitrogen and storage

(b) modified freezing in a controlled rate freezer before storage in the LN2 tank. As a result of cryopreservation, KOPRI AnM0046 survived after both uncontrolled direct freezing and modified freezing.