Journal of Agricultural Science; Vol. 12, No. 7; 2020 ISSN 1916-9752 E-ISSN 1916-9760 Published by Canadian Center of Science and Education 75 Cryopreservation of Chlorella vulgaris Using Different Cryoprotectant Agents Helder Rodrigues da Silva 1 , Francino Costa Palhares da Silva 1 , Cassio Egidio Cavenaghi Prete 2 , Rodrigo Thibes Hoshino 2 , Ricardo Tadeu de Faria 2 , Mario Sergio Mantovani 3 & Carmen Luisa Barbosa Guedes 1 1 Postgraduate Program in Bioenergy, Center for Exact Sciences, Universidade Estadual de Londrina, Londrina, Paraná, Brazil 2 Center for Agricultural Sciences, Department of Agricultural Sciences, Universidade Estadual de Londrina, Londrina, Paraná, Brazil 3 Center for Biological Sciences, Department of General Biology, Universidade Estadual de Londrina, Londrina, Paraná, Brazil Correspondence: Helder Rodrigues da Silva, Postgraduate Program in Bioenergy, Center for Exact Sciences, Universidade Estadual de Londrina, Paraná, Brazil. Tel: 55-433-371-5453. E-mail: heldersilva@uel.br Received: March 1, 2020 Accepted: April 7, 2020 Online Published: June 15, 2020 doi:10.5539/jas.v12n7p75 URL: https://doi.org/10.5539/jas.v12n7p75 The research is financed by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil). Abstract The objective of this study is to evaluate the cryopreservation of Chlorella vulgaris using different substances. The C. vulgaris was cultured in medium MH, the microalgae were grown under a 12:12 h light: dark photoperiod, illumination with 40 W led lamps, and a controlled temperature of 28±1 ºC. C.vulgaris was cultured for 15 days and the culture was aliquoted into 3-mL cryogenic tubes. The 3-mL aliquot was centrifuged, the supernatant was discarded, and the pellet was resuspended in different cryoprotectant solutions, T1-PVS1, T2-PVS2, T3-PVS2 (1% phloroglucinol), T4 (2 M glycerol), and T5 (5% methanol). The samples were rapidly frozen in liquid nitrogen (-196 °C) and analyzed after 15, 150, and 300 days of freezing. Cell viability was determined in cultures grown for 20 days. The only effective treatment was T5, which promoted the growth of thawed cultures in both solid and liquid media. After 15 days of freezing in liquid nitrogen and 20 days of culture growth, the number of viable and nonviable cells was 3.42±0.72 × 10 7 and 0.06±0.009 × 10 7 , respectively, and viability was 98.2%. Similar values were obtained after 150 and 300 days of freezing: 2.17±0.15 × 10 7 and 2.35±0.18 × 10 7 viable cells, 0.05±0.02 × 10 7 and 0.10±0.02 × 10 7 nonviable cells, and viability of 97.6% and 95.8%, respectively. The cryopreservation protocol for microalgae C. vulgaris using 5% methanol was effective; therefore, it is possible to maintain this strain under axenic conditions in liquid nitrogen for long periods. Keywords: liquid nitrogen, conservation, microorganism, microalgae, biotechnology, bioenergy, biofuels 1. Introduction Microalgae are photosynthetic microorganisms that can produce different metabolites, including lipids, proteins, carbohydrates, and pigments. A study has demonstrated the potential of microalgae as a raw material for producing biofuels, nutraceuticals, cosmetics and pharmaceuticals, bioestmulants and biofertilizer for agricultural and for other applications (Andrade et al., 2014; Richmond, 2004; Silva et al., 2016). One of the challenges in microalgae production is the maintenance of strains in the laboratory. The methodology most commonly used for growing microalgae is subculturing samples isolated in solid or liquid medium at low temperatures and low-light conditions to minimize biological activity and growth (Lorenz, Friedl, & Day, 2005). This procedure requires the long-term and intensive use of labor and materials and presents the risk of contamination and changes in genetic stability because phenotypic variations occur after successive subculturing over time (Day et al., 2005). Several studies developed new techniques to preserve these microorganisms and reduce cost and labor, with particular emphasis on the development of effective cryopreservation techniques (Lourenço, 2006).