Tools for biotechnological studies of the freshwater alga Nannochloropsis limnetica: antibiotic resistance and protoplast production

Algae of the genus Nannochloropsis are attractive organisms for use in biotechnology due to their high lipid content. Genetic manipulation of marine Nannochloropsis species has already been reported; however, tools have not yet been developed to transform Nannochloropsis limnetica, the only known freshwater species of this genus. To establish N. limnetica as a model laboratory strain, we first tested the effects of 11 different antibiotics on growth of N. limnetica and the marine species N. oceanica and N. gaditana. These three microalgae responded very differently to antibiotic treatments, both in liquid cultures and on agar plates. In general, N. limnetica exhibited a much higher sensitivity to antibiotics than the marine strains, thus offering the potential for a large set of antibiotic resistance genes that may be applicable as artificial selection markers after transformation. We also developed a simple protocol using lysozyme to obtain high yields of viable N. limnetica protoplasts, as confirmed by flow cytometry and electron microscopy.