Abstract
The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO) were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage), the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components) and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage). The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3–50 µm), a variable that can affect cryopreservation success. The collective improvement of each of these parameters yielded a cryopreservation protocol that can be applied to a broad range of microalgae.
Highlights
Microalgal biotechnologies are rapidly being developed for commercial exploitation of a range of products including health supplements, animal feeds, biofuels and chemical feed stocks [1]
It is estimated that approximately 350,000 microalgae species [2] may exist in a diverse range of habitats, ranging from fresh to saline water sources and from arctic conditions to thermal springs [3]
This biodiversity provides an excellent basis for future microalgae breeding programs which depend on extensive culture collections
Summary
Microalgal biotechnologies are rapidly being developed for commercial exploitation of a range of products including health supplements, animal feeds, biofuels and chemical feed stocks [1]. It is estimated that approximately 350,000 microalgae species [2] may exist in a diverse range of habitats, ranging from fresh to saline water sources and from arctic conditions to thermal springs [3]. This biodiversity provides an excellent basis for future microalgae breeding programs which depend on extensive culture collections. There is a need for improved cryopreservation techniques. The aim of this paper is to improve our understanding of the key steps in the cryopreservation process and to optimize each of these
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