DEVELOPMENT ON A CRYOPRESERVATION PROTOCOL OF MICROCYSTIS AERUGINOSA USING PENETRATING AND NON-PENETRATING CRYOPROTECTANTS

Microcystis aeruginosa Kütz is a freshwater bloom-forming cyanobacterium that is found all over the world. Owing to their adverse effects on water quality and human health, these blooms in water of reservoirs and recreational water systems are a cause of great concern, M. aeruginosa became to a popular research object. Previous studies have revealed significant differences between the physiological parameters and stress responses of individual cells in lab and wild type colonial of Microcystis in fields. Therefore, to avoid the loss of natural characteristics, an efficient technology or method would have to be used for preservation of M. aeruginosa. Cryopreservation is a useful method to preserve cells and organs for a long time in lab. It was thus used on M. aeruginosa in order to keep the physiological characteristics stable at long-term studies. In the present study, cryopreservation of three strains M. aeruginosa (PCC7806, FACHB-905 and FACHB-942) was accomplished successfully. Combination of three penetrating cryoprotective additives (CPA) (methanol, dimethylsulphoxide (Me2SO), glycerol) and non-penetrating polyvinylpyrrolidone (PVP) could improve viability almost 20% than only using the same penetrating CPA. The optimal protocol of cryopreservation was screened from CPAs above and four concentrations of each, two cooling rates (-1 /min ℃ n and -0.5℃/min), and the first-step temperature degreed (-30℃, -40℃ and -80℃) of traditional two step methods. In addition, cell viability was determined by performing flow cytometry with fluorescein diacetate (FDA). The results showed that an optimal cooling protocol was at cooling rate of 0.5℃/min to -40℃ and combination of penetrating CPA (Me2SO, 5%) and non-penetrating CPA (PVP, 30%). The ratios of viable cells in the FACHB-942, PCC-7806 and FACHB-905 were approximately (62.72 ± 0.15)%, (66.3 ± 0.65)%, and (71.69 ± 0.39)%, respectively. However, the ratios of viable cells in these strains were all only about 45%. At this protocol of cryopreservation, physiological parameters of three strains M. aeruginosa such as PSII, the cells pigments and specific growth rates were analyzed before preservation and after thaw, results about the comparison showed that M. aeruginosa could keep its physiological characteristics stable.