Establishing and validating axenic cultures of the microalga Haematococcus lacustris (Chlorophyceae)

Although many isolation techniques for algaeaxenic cultures of algae are known, successful isolation approaches are species and contaminant-specific. The commercially important alga Haematococcus lacustris has been intensively studied due to its natural production of the high-value carotenoid astaxanthin, yet there are no reports of axenic H. lacustris cultures. In this article, we describe the successful isolation of axenic H. lacustris, originally isolated in non-axenic form from its natural habitat and adopted to be used daily in vitro, in our laboratory. We verify the absence of bacteria and identify the bacterial communities in the non-axenic algal culture. using PCR amplification with selected universal primers for 16S rRNA gene amplification in the gDNA. Amplification of the 16S rRNA gene of the non-axenic culture showed the presence of bacteria with high identity to Massilia, Blastococcus and Deinococcus species. The bacteria which were identified in our strain further expand our knowledge of bacterial communities on algae, and because we established axenic H. lacustris culture, testing their effect on growth or astaxanthin accumulation in controlled co-cultures is now available. The isolation technique described herein can be applied to other green algal species to eliminate bacteria, and the selected primers can be used to verify the axenic nature of other green algal cultures. The resulting axenic culture is required for molecular genetics research and will be very valuable for establishing heterotrophic growth in laboratory or industrial bioreactors.