Microalgae and cyanobacteria provide the basis of the food chain in most aquatic environments. Currently described microalgal species are around 40.000-50,000, and it is estimated that the total species may be approximately one million. In aquaculture, filter feeders represent a quarter of the world production. Bivalves, fish and early life stages of many aquatic organisms, consume microalgae as their main nutrient source. Thus, hatcheries for these organisms rely on a steady supply of microalgae. Additionally, microalgae are an important feedstock for marine biotechnology products, a sector estimated to be worth over $3 billion dollars. Microalgal strains have been traditionally maintained as live cultures. Due to the time, cost and risk of maintaining these live cultures, as well as the routine loss of traits, only a few thousand strains are maintained in culture collections. To overcome these problems, culture collections have turned to cryopreservation to maintain strains that are less frequently used. The protocols used to this end, have been developed from those used for animal sperm preservation, and are not universally suited for microalgae.
Most microalgal strains preserved are obtained from active cultures and preserved in 0.5 to 1.0 mL containers. This small volume is enough to start cultures in small volumes (1-20 mL) , and it is useful for the preservation of the genetic material. The recovery of these strains for aquaculture use, requires careful planning, as it will be necessary to expand the cultures from this small volume to those needed for hatchery operations. As there is no standardization regarding cell counts or volume and cryoprotectant dose, the time to recover strains from cryopreservation may vary amongst preparations.
In this work, we explored the possibility of using high throughput cryopreservation, with standardized, low-cost containers (0.5 mL French straws), that can be labeled, packed and frozen at the rate of tens of thousands of samples per hour, in a standardized, repeatable way. Microalgal samples of different species, including Tetraselmis chuii, Chaetoceros muelleri and Tisochrysis lutea, were cryopreserved at the culture density, and after concentration to 108-109 cells mL-1. The concentration of the cells was done by two methods: centrifugation and water removal trough superabsorbent polymers, and combinations of these two methods. The results show that microalgae can be successfully recovered after cryopreservation under standardized, high throughput conditions. The efficiency of the superabsorbent polymers is affected by the salinity of the culture media. Sodium-based polymers are more affected by salinity than the potassium-based polymers. Cryopreserving concentrated samples, a 0.5 mL straw can be used to start cultures 50-500 times larger than samples without concentration, saving weeks of wait for the culture to grow. Concentrating algae for cryopreservation maximize the use of available resources, reduce space, and labor, and preserve higher density biomass for research, conservation and animal feeding.