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 highthroughput 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 rapidgrowth 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 to be invaluable for the selective screening of microalgae.