In this issue, we recommend an article by Professor Wei Dong's team from the School of Food Science and Engineering at South China University of Technology, published in the international journal Algal Research 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。 This study utilized an integrated system of atmospheric pressure room temperature plasma mutagenesis (ARTP) and high-throughput microbial droplet culture (MMC) to rapidly screen for high protein mutant strains of Chlorella pyrenoidosa, providing promising candidate bacteria for heterotrophic fermentation to produce alternative proteins.

With the growth of the global population, the demand for food is also increasing, which exacerbates the need to find new sources of protein amidst the reduction of arable land and ethical concerns over traditional meat production. Microalgae, as a promising alternative protein source, are rich in protein, amino acids, polyunsaturated fatty acids (PUFAs), vitamins, and minerals. However, under heterotrophic cultivation conditions, microalgae typically exhibit lower protein content (<40% dry weight) due to reduced protein synthesis dependent on photosynthesis, limiting their potential as an alternative protein source. To overcome these challenges, developing novel microalgae strains with natural high protein content is crucial for efficient large-scale protein production through heterotrophic fermentation.

Researchers used ARTP mutagenesis to obtain the starting strain of Chlorella pyrenoidosa A4-1, and conducted a new round of ARTP mutagenesis. After 15 seconds of irradiation, the strain was transferred to shake flask culture. Dilute the cells in logarithmic growth phase to an OD450nm value of 0.6-0.8 and transfer them to the MMC system for further cultivation.

In the MMC system, an initial 50 droplets are generated, with each generation running for 24-46 hours. The absorbance value of the droplets at 450 nm is detected in real-time to characterize the growth of the algal strain. After three rounds of cultivation, the droplet with good growth (droplet 28) was selected. The entire experimental process only took 116 hours, which is a significant improvement compared to the traditional tablet system (Figure 2A). The droplets were subjected to monoclonal sorting to obtain 23 strains of bacteria. Among them, 4 strains (MMC-1, 7, 8, and 11) with higher growth rates or biomass concentrations were selected for subsequent analysis (Figure 2B).

The cell growth and biomass yield of four selected mutants (MMC-1, 7, 8, 11) cultured in 250 mL shake flasks are shown in Figure 3A. The growth pattern of all mutants is similar to that of the A4-1 algal strain, experiencing an initial lag phase and then rapidly entering an exponential growth phase. MMC-7 reached the highest biomass concentration of 8.21 g/L, which was 8.49% higher than the 7.57 g/L of A4-1 strain (p<0.05). At the same time, all four mutants visually exhibited a golden color similar to the A4-1 algal strain, with no detected chlorophyll b and chlorophyll a accounting for only 1% of the wild-type (WT) (Figure 3B).

Biochemical analysis showed that protein content increased by 12% to 40% in the four mutants (Figure 4). The MMC-8 mutant showed the highest protein content (63.26% dry weight) and low starch content (8.59% dry weight), with an increase of 40.11% and a decrease of 56.24% compared to the original algal strain A4-1, respectively. In addition, MMC-8 also performs well in protein quality, with higher amino acid content (44.35% dry weight) and score (95) than A4-1.

This study demonstrates the potential of the ARTP-MMC system as a powerful high-throughput screening platform, which not only improves protein synthesis efficiency but also reduces starch content, tilting carbon allocation towards protein synthesis. This is of great significance for improving the efficiency and sustainability of biological protein production.

Paper link: https://doi.org/10.1016/j.algal.2024.103509