Breeding Chlorophyll-Deficient Mutants of Chlorella vulgaris to Enhance Consumer Acceptance

The use of microalgae as a food source is limited by consumers' dislike of their organoleptic traits, primarily the intense green color and bitter taste associated with high chlorophyll content. The eukaryotic microalgae <i>Chlorella vulgaris</i> can grow under heterotrophic conditions, providing the opportunity to cultivate chlorophyll-less strains. In this work we applied random mutagenesis for breeding chlorophyll-deficient <i>C. vulgaris</i> 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 chlorophyll was observed in M11 when grown under light. Under heterotrophic and mixotrophic growth conditions, both mutants were chlorophyll-deficient while biomass productivity, protein content, and amino acid composition remained similar to 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 mutants. Our work demonstrates how a simple approach using UV mutagenesis and visual screening can provide novel strains of <i>C. vulgaris</i> with traits for improved consumer acceptance, without compromising the use of the algae biomass as a protein-rich food source.