Efficient heterotrophic cultivation of Chlamydomonas reinhardtii

Abstract

With a unique cell structure and clear genetic background, Chlamydomonas reinhardtii has become a model species for fundamental research on microalgae and lower plants. Genetic transformations in the nucleus and chloroplast and mitochondrial genomes of C. reinhardtii have been successfully developed. However, research of C. reinhardtii has mainly focused on physiology and molecular genetics and few studies investigating cultivation methods have been performed. Small scales, slow growth, and low yields from conventional photoautotrophic and mixotrophic cultures severely limit the further development and application of C. reinhardtii. This study sought to optimize and scale up the heterotrophic cultivation process for C. reinhardtii. Critical parameters in 500-mL shake flasks were optimized as follows: liquid volume of 150 mL, initial inoculation of 0.08 g L−1, growth temperature of 30 °C, and shaking speed of 150 rpm. In addition, this study confirmed that, although C. reinhardtii can assimilate acetic acid for heterotrophic growth (optimum initial concentration of 80 mM), neither glucose nor glycerol can be the only carbon source. Nitrate, ammonia, and urea can be used as nitrogen sources for heterotrophic culture, while the optimal carbon and nitrogen ratio for heterotrophic culture was 40:1. Under optimal conditions, the C. reinhardtii biomass increased from 0.457 to 1.32 g L−1. Furthermore, for the first time, heterotrophic cultures of C. reinhardtii were scaled up to 5-L and 50-L fermenters, in which the cell density reached 25.44 g L−1 after 237 h, which is 2.82-fold higher than previously reported.