Cryopreservation of Chlorella vulgaris Using Different Cryoprotectant Agents

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 and 0.06±0.009 × 10, respectively, and viability was 98.2%. Similar values were obtained after 150 and 300 days of freezing: 2.17±0.15 × 10 and 2.35±0.18 × 10 viable cells, 0.05±0.02 × 10 and 0.10±0.02 × 10 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.


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 .
Cryopreservation is a methodology used to preserve microorganisms at ultra-low temperatures (less than -130 °C) and allows their growth after thawing (Day & Brand, 2005;Tessarolli, Day, & Vieira, 2017).
The advantages of cryopreservation are long-term stability, reduced material costs, protection against genetic drift and contamination, and reduced cost of long-term maintenance (Fernandes et al., 2019;Prakash, Nimonkar, & Shouche, 2013;Tessarolli et al., 2017). Although the procedures for cryopreserving microorganisms are usually simple, complex cell culture protocols involving multi-stage freezing at specific cooling rates and storage in liquid nitrogen are required for microalgae (Day & Harding, 2008).
Another determinant factor in the success of cryopreservation is the composition of the freezing medium, in which reagents and concentrations depend on the sensitivity of each strain (Hubalek, 2003). In view of the heterogeneity of microalgae, many strains require specific protocols and standards, and many species do not adapt to the freezing technique (Taylor & Fletcher, 1998).
A plant vitrification solution (PVS) is used for cryopreserving plant tissues, including embryos, protocorms, pollen, seeds, and cell suspensions (Sakai & Engelmann, 2007). Nonetheless, no studies have evaluated the potential use of PVS for microalgae.
Given the complexity of the protocols and the need for specific equipment, the objective of this study is to evaluate the effectiveness of different cryoprotective agents in preserving microalgae Chlorella vulgaris.

Microalgae and Culture Conditions
The experiments were carried out in the Laboratory of Chemistry of Biomass, Biofuels, and Bioenergy (Laboratório de Química da Biomassa, Biocombustíveis e Bioenergia-LAQUIBIO) and in the Laboratory of Crop Science of the Universidade Estadual de Londrina-UEL, Londrina, Paraná, Brazil.
A strain of the microalga Chlorella vulgaris was obtained from the Laboratory of Crop Science of UEL and was maintained in solid and liquid culture media under axenic conditions. Subculture in liquid medium under aseptic conditions was performed to obtain the stock culture. MH medium with the following composition was used: 0.57 g L -1 MAP, 0.075 g L -1 CaCl 2 , 0.36 g L -1 KNO 3 , 0.225 g L -1 MgSO 4 , 0.09 g L -1 YaraVita Rexolin BRA® (11.6% K 2 O, 1.28% S, 0.86% Mg, 2.1% B, 0.36% Cu, 2.66% Fe, 2.48% Mn, 0.036 % Mo, and 3.38% Zn) (Silva, 2016). The pH was adjusted to 7.0 using 1 M KOH.
The stock culture was maintained in a growth chamber for 15 days in 1-L Erlenmeyer flasks containing 500 mL of MH medium under a 12:12 h light: dark photoperiod, illumination with 40 W led lamps, and controlled temperature of 28±1 °C.

Cryopreservation Protocols
After initial culturing, the stock was aliquoted into 3-mL cryogenic tubes. A 3-mL aliquot was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in different freezing media.
The pellets were resuspended in 1.4 mL of the freezing medium at 4 °C, kept in an ice bath for 20 min, and subjected to rapid freezing in liquid nitrogen (-196 °C). Centrifugations and the addition of the cryoprotective solutions were performed in a laminar flow chamber under aseptic conditions. All assays were performed in triplicate.

Thawing and Recovery Growth
Thawing was performed after 15, 150, and 300 days of freezing. The tubes were removed from the liquid nitrogen and thawed by manual stirring at room temperature.
Test tubes containing 2 mL of sterile MH medium were inoculated with 200 μL of the thawed suspension, without performing centrifugation and washing procedures for removing the cryoprotectants.
In the first thawing time point (15 days), 100 μL of the thawed suspension was dispersed in solid MH medium (1% agar) using a Drigalski loop to assess growth on semisolid medium. At 150 days of freezing, Erlenmeyer flasks containing 100 mL of MH medium were inoculated with 800 μL of the thawed suspension. jas.ccsenet.
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Several factors may affect the efficiency of cryopreservation protocols, including cell density of stock cultures, number of centrifugation steps, light intensity during culturing, composition of the freezing medium, and the interactions of cryoprotectants with different species of microorganisms (Canavate & Lubián, 1995).
In the scientific literature the cryoprotective potential of several substances was evaluated. The most effective substances were DMSO, methanol, ethylene glycol, and propylene glycol, and the least effective were glycerol, polyethylene glycol, PVP, and sucrose. DMSO is widely used as a freezing medium but is toxic to most microorganisms (Hubalek, 2003); therefore, many studies seek to define optimal combinations and concentrations of cryoprotectants. Bui et al. (2013) demonstrated the synergistic effect of DMSO combined with 200 mM sucrose. The optimal concentration of DMSO was 6.5%, and higher concentrations reduced cell viability in Palmellopsis sp. and Chlamydomonas sp. For Chlorella vulgaris, Nannochloropsis oculata, and Tetraselmis tetrathele, 2.5% or 5.0% DMSO in isolation has no cryoprotective effect. However, the combination of 5% DMSO and 5% ethylene glycol resulted in survival of 10% for Chlorella vulgaris, and survival increased to approximately 50% when 5% proline was added to the freezing medium (Nakanishi, Deuchi, & Kuwano, 2012).
Another critical factor to consider is the cooling rate. The above studies used slow cooling in two stages, in which, after adding the cryoprotectants, the tubes were transferred to freezers and frozen at a cooling rate at -1 °C min -1 until they reached temperatures of -40 °C to -80 °C. The tubes remained at this temperature range for 4 to 16 h and were then immersed in liquid nitrogen. Buhmann, Day, and Kroth (2013) found that the cryopreservation of Planothidium frequentissimum using 5% DMSO was feasible by pre-freezing at -40 °C at a controlled cooling rate of 1 °C min -1 and maintenance in the dark for 48 hours in the post-thawing recovery phase to reduce photooxidative stress. Morschett, Reich, Wiechert, and Oldiges (2016) reported that direct freezing in liquid nitrogen reduced the viability of Chlorella vulgaris maintained in 10% DMSO, 10% ethylene glycol, and 10% L-proline.
Another factor that may have affected the effectiveness of PVS is the step of washing and removing the cryoprotective agents. The steps performed after thawing included centrifuging the tubes, discarding the supernatants, and resuspending the pellet in culture medium or saline before inoculation in the recovery medium. Although decreasing the number of centrifugations increases cell viability by reducing mechanical damage (Bui et al., 2013), skipping the centrifugation washing step may be detrimental because of the high toxicity of the cryoprotectants at room temperature, even at low concentrations, diluted in the recovery medium.
These results indicate the advantages of T5 because the cryopreservation protocol used both before and after freezing is simple, and some steps are unnecessary, including controlled cooling, culture maintenance at pre-freezing temperatures, centrifugation to remove the cryoprotective agents, and recovery growth under low-light conditions. These characteristics make the T5 protocol simple, fast, and effective for preserving Chlorella vulgaris. Scarbrough and Wirschell (2016) have shown that 5% methanol is better than commercial kits for preserving Chlamydomonas reinhardtii because the thawed culture can be directly inoculated in semisolid agar or liquid medium under aeration. Furthermore, aeration was essential to guarantee cell viability during culture recovery in liquid medium.