High-density heterotrophic cultivation of a cell-wall-deficient Chlamydomonas reinhardtii strain by fed-batch...

Chlamydomonas reinhardtii CW15, a cell-wall-deficient strain, is a widely used chassis in microalgal genetic engineering. However, its low cell density under autotrophic and mixotrophic cultivation restricts industrial scalability. This study aimed to establish a cost-effective, high-density heterotrophic cultivation process for C. reinhardtii CW15. Initially, an optimized AP medium (Tris-free TAP with K₂HPO₄ as the sole phosphorus source) significantly reduced medium costs while achieving a cell density of 0.446 g/L. Subsequently, a fed-batch process in a 5 L fermentor yielded a record cell density of 22.1 g/L (growth rate: 94.6 mg/L·h) at 233 h by optimizing nutrient supplementation. Scaling up to a 50 L fermentor revealed challenges in ammonia volatilization and salinity accumulation during sterilization. These were resolved by substituting the nitrogen source with ammonia water and supplementing ammonium acetate as a dual nitrogen and partial carbon source, ultimately achieving 30.2 g/L (140 mg/L·h) after 215 h-the highest reported density for cell-wall-deficient C. reinhardtii. This work provides an efficient and scalable cultivation strategy, facilitating its industrial and biotechnological applications.