Short-term deleterious effects of standard isolation and cultivation methods on new tropical freshwater microalgae strains

Algae with potential biotechnological applications in different industries are commonly isolate 5 from the environment in order to obtain pure (axenic) stocks that can be safely stored for long 6 periods of time. To obtain axenic cultures, antibiotics are frequently used, and cryopreservation 7 is applied to keep standing stocks. However, many of these now standard methods were 8 developed using strains coming from pristine-to-low intervened environments and cold-to- 9 temperate regions. Thus, it is still not well understand the potential effect of said methods on the 10 life cycle and biochemical profile of algae isolates from hiper-eutrophic and constant high- 11 temperature tropical regions, which could potentially render them unsuitable for their intended 12 biotechnological application. In this study, we did a genetic characterization (18s mDNA) and 13 evaluated the effect of purification (use of the antibiotic chloramphenicol, CPA) and 14 cryopreservation (dimethyl sulfoxide (DMSO)-sucrose mix and Glycerol) on the growth rate and 15 lipid content of four new tropical freshwater algal isolates: Chorella sp. M2, Chlorella sp. M6, 16 Scenedesmus sp. R3, and Coelastrella sp. A2, from the Ecuadorian coast. The genetic 17 characterization showed a clear discrimination between strains. All strains cultured with CPA 18 had a lower growth rate. After cryopreservation Coelastrella sp. A2 did not grow with both 19 methods; Chorella sp. M2, Chlorella sp. M6 and Scenedesmus sp. R3 presented no significant 20 difference in growth rate between the cryopreservants. A significantly higher lipid content was 21 observed in biomass cryopreserved with glycerol in relation to DMSO-sucrose, with Chorella sp. 22 M2 and Chlorella sp. M6 having twice as much in the first treatment. The results highlight the 23 relevance of choosing an appropriate method for storage as the materials used can affect the 3 1 biological performance of different tropical species, although is still to determine if the effects 2 observed here are long lasting in subsequent cultures of these algae.