Cryopreservation of the Chlorophyll d-Containing Cyanobacterium Acaryochloris Marina

Abstract

Cryopreservation of a prokaryotic alga, Acaryochloris marina, which was first isolated as a minor symbiont from a colonial ascidian, was investigated. This alga, possessing chlorophyll d as the major photosynthetic pigment, is an important organism for elucidating various biological questions such as the mechanism of chlorophyll diversity and the evolution of photosynthesis. However, the proportion of photosynthetic pigments changes on serial subcultures on maintenance under a fluorescent lamp, a light condition that differs from that of the original habitat. Cryopreservation has the capacity to ensure the maintenance of the physiological and genetic stability of A. marina. By placing samples in a styrene foam container in a deep freezer, cooling rate of –2 °C·min-1 was obtained for pre-freezing to –80 °C prior to plunging into liquid nitrogen. Amongst the three distinct cryoprotectants employed, [dimethyl sulfoxide (DMSO, 10% v/v), methanol (10% v/v), and glycerol (10% v/v)], DMSO was the most effective since cells preserved in DMSO for one month in liquid nitrogen or at –80 °C in a deep freezer, started to grow 200 h after inoculation at 20 °C under a fluorescent lamp. In contrast, no cell growth was observed when cells were preserved employing methanol, and growth of contaminant bacteria was observed when cells were preserved in glycerol. The optimum concentration of DMSO was 10%. Other concentrations of DMSO resulted in an extended lag period at 5% and 20% DMSO, and growth was completely inhibited at 40% DMSO.