Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains

Isolation of High-Lipid Tetraselmis suecica Strains Following Repeated UV-C Mutagenesis, FACS, and High-Throughput Growth Selection

“…However, although the frequency of mutants increases among survivors at high dosages, so does damage to the genetic background which reduces clone recovery and fitness. Therefore, two different UV-C dosages were chosen to either increase mutation probability (>98 % lethal dosage) or increase recovery of a high number of clones (50 % lethal dosage) with less genetic background damage, a survival rating similarly applied in other microalgae mutation studies [19,22]. The recovery of UV-C-exposed cells in liquid media was more effective, as survival rates in 96 well-plates were higher compared to solid media agar plates.…”

“…The use of Nile red as a fluorescence probe for neutral lipid detection and quantification in microalgae has been well documented [1,21,22,24,23,33,34]; a strong correlation between lipid content and fluorescence intensity has already been established [34]. The addition of solvents such as acetone and DMSO has been shown to improve the transition of the dye into lipids, although their efficacy varies between species, depending on the characteristics of the individual algae species [34].…”

“…The first kill curve was obtained by plating 200 μL aliquots of 1/1000 diluted mutagenized cells onto a 1 % agar plate containing F medium in artificial seawater, which were then allowed to grow for 3 weeks before algal colonies from control and UV-exposed cells were counted. The second kill curve was carried out in 96-well plates inoculated with mutagenized cells after serial dilution down to 1 cell per well [22]. Wells with surviving microalgae that multiplied were then counted after 3 weeks of growth.…”

“…Mutation studies involving ultraviolet (UV), ethyl methane sulfonate (EMS), and nitrosomethylguanidine (NTG) have been performed on a range of microalgal species (Phaeodactylum tricornutum, Pavlova lutheri, Nannochloropsis oculata, Haematococcus pluvialis, Schizochytrium sp., Chlorella sorokiana, Scenedesmus obliquus, Isochrysis galbana, Dunaliella salina) to produce mutants that exhibited increased/ modified lipid content and growth rate [14][15][16][17][18][19][20][21][22][23]. Many of these studies, however, rely upon time-consuming techniques like metabolite antibiotic response to select for mutants and lipid extraction techniques to analyze their lipid content.…”

“…Automated fluorescence-assisted cell sorting (FACS) can be more efficient than a microplate readerbased selection, as much larger populations of single cells can be handled, resulting in the recovery of a high number of candidate cells with the desired lipid content. The use of FACS in combination with mutagenesis has been described in mutationselection studies that produced high-lipid content strains of I. galbana [23] and carotenoid-hyperproducing D. salina strains [22]. Even without mutagenesis, the use of FACS has been successful in generating higher-lipid content strains of Nannochloropsis sp.…”