Breeding Chlorophyll-Deficient Mutants of <em>Chlorella vulgaris </em>to Enhance Consumer Acceptance

The prevalence of microalgae as a food source is challenged by the consumers disliking the organoleptic traits mainly due to the intense green color and bitter taste associated with the high chlorophyll content. The eukaryotic microalgae Chlorella vulgaris can grow under heterotrophic conditions providing the opportunity to cultivate chlorophyll-less strains. In this work we applied random mutagenesis for breeding chlorophyll deficient C. vulgaris strains. Wild type strain was UVC-radiated, and 12 colonies with changed pigmentation were selected. Based on phenotypic stability two mutants, M6 and M11, were selected for characterization of growth, pigment- and biomass accumulation. Cultivation under photo-, mixo-, and heterotrophic conditions revealed distinct phenotypes for the two mutants. M6 remained chlorophyll deficient in all cultivation conditions tested, while limited amounts of chlorophyll were observed in M11 when grown in light. At heterotrophic and mixotrophic growth conditions, both mutants were chlorophyll defi-cient while biomass productivity and protein content and amino acid composition were not significantly different from wild type. Characterization of the cellular ultrastructure of the wild type and mutants using cryo Focused Ion-Beam Scanning Electron Microscopy revealed that functional chloroplasts and thylakoid membranes were absent in the mu-tants. Our work demonstrates how a simple approach using UV mutagenesis and visual screening can provide novel strains of C. vulgaris with traits for improved consumer acceptance, without compromising the use of the algae biomass as a protein-rich food source.