Rapid screening of high-protein Auxenochlorella pyrenoidosa mutant by an integrated system of atmospheric and room temperature plasma mutagenesis and high-throughput microbial microdroplet culture

Microalgae cultivated heterotrophically often exhibit high biomass concentration but low protein content. An integrated system combining atmospheric and room temperature plasma (ARTP) mutagenesis with high-throughput microbial microdroplet culture (MMC) was introduced to rapidly breed high-protein strains of Auxenochlorella pyrenoidosa for heterotrophic fermentation. Utilizing the previously identified A4–1 mutant strain as original strain, ARTP-induced mutants were screened through MMC, prioritizing mutants with rapid-growth and high-protein via subsequent well-plate and shake flask cultivation based on evaluation of growth and biochemical composition. The MMC-8 mutant exhibited the highest protein content (63.26 % dry weight, DW) and the lowest starch content (8.59 % DW), representing a 40.11 % protein increase and a 56.24 % starch decrease relative to A4–1. MMC-8 also demonstrated superior protein quality, with a higher amino acid content (44.35 % DW) and score (95) than the A4–1. These attributes establish MMC-8 as a promising candidate for alternative protein production through heterotrophic fermentation, while the ARTP-MMC system was proved be invaluable for the selective screening of microalgae.