Application and theory of algal cryopreservation
Brand, Jerry J.; Diller, Kenneth R.
Cryopreservation is a practical method for maintaining a broad range of microalgae over long periods of time. The Culture Collection of Algae at the University of Texas at Austin (UTEX) currently successfully maintains over 1300 strains of algae under cryopreservation, although many other strains at UTEX cannot yet be cryopreserved or else survive with very low viability. The ability of a culture of microalgae to survive freezing and thawing is strongly dependent on the conditions under which it is frozen, including the nature and concentration of a cryoprotective agent added to the algal culture prior to freezing. Experiments using the unicellular microalga Chlorococcum texanum Archibald et Bold (UTEX #1788), demonstrate that methanol is transported across the plasma membrane faster than water, while dimethyl sulfoxide is transported more slowly than water. These differences cause very different volumetric excursions of algal cells in response to the added cryoprotective agent. This may help explain why different algae respond differently to methanol and dimethyl sulfoxide, although both are widely used as cryoprotective agents. An often overlooked parameter during cryopreservation of algae is the culture density at the time of freezing. Studies using Chlamydomonas reinhardtii Dangeard (UTEX #89) demonstrate that low culture density during freezing is important for high viability after thawing from liquid nitrogen. The low viability of C. reinhardtii cultures cryopreserved at high cell density is caused by an unidentified water-soluble substance that is released into the medium from cells that are killed during cryopreservation. At sufficiently high concentration the substance is lethal to other cells in the medium when the cryopreserved culture is thawed. The inhibitory substance is small, water soluble and heat-stable, but its release from cells is prevented by heat-denaturing the algal cells prior to their freezing and thawing.