Is axenicity crucial to cryopreserve microalgae?

Large culture collections of microalgae and cyanobacteria such as the Coimbra Collection of Algae (ACOI) hold unialgal cultures consisting of a population of cells/colonies of a certain species. These cultures are usually non-axenic, as other organisms such as bacteria and microfungi are also present in culture due to co-isolation. Attention has been recently given to partner organisms since studies indicate that some bacteria are important for nutrient uptake of the algal cells, acting as simbionts. Despite this benign effect in the actively growing cultures, when cryopreservation is applied for inactive-stage storage, these organisms may recover faster than the algae, thus affecting their recovery and the viability assessments. In this study, a set of mucilaginous ACOI microalgae were selected, cell features known for their relevance in cryopreservation success were recorded and simple two-step cryopreservation tests were applied. Thawed samples were transferred to fresh culture medium for recovery. Viability was assessed and partner organism proliferation (pop) was recorded. Results were analyzed by t-tests. Statistical models allowed us to support the known tendency for small, unicellular algae with no outer structures to be successfully cryopreserved and the negative effect of vacuoles in the cell prior to cryopreservation. On average cryopreservation with MeOH or Me2SO led to the recovery of nearly half the cells. It was found that the cryoprotection step with MeOH is when pop is triggered and that the use of Me2SO can prevent this effect. Progress on understanding the cultured consortia will assist the improvement of cryopreservation and research using microalgal cultures.