Metabolic Response of Extremophile Halochlorella Rubescens on Carbohydrate Biosynthesis Cultivated Under High Carbon-Rich Condition

Microalgae cultivation under elevated carbon dioxide (CO₂) concentrations may negatively affect biomass production and intracellular carbohydrate biosynthesis, posing a major challenge for large-scale industrial applications. Despite the growing interest in carbohydrate-based bioproducts derived from microalgae, the underlying mechanisms regulating carbohydrate accumulation under high CO₂ conditions remain insufficiently understood. This study investigated the effects of CO₂ concentrations ranging from 0.04% to 25% on the growth performance, morphological characteristics, and carbohydrate productivity of Halochlorella rubescens. The highest biomass concentration (0.65 g L⁻¹) and carbohydrate productivity (16.34 mg L⁻¹ d⁻¹) were achieved at 5% CO₂, indicating that moderate CO₂ enrichment enhances carbon assimilation and carbohydrate accumulation. However, exposure to higher CO₂ levels altered cellular morphology and influenced the expression of key proteins involved in carbohydrate biosynthesis. Transcriptomic and proteomic analyses revealed significant enrichment of genes associated with carbon fixation, photosynthesis, starch synthesis, and carbohydrate metabolic pathways under elevated CO₂ conditions. Notably, the upregulation of photosynthesis-related proteins (ATP synthase, Photosystems I and II, and chlorophyll a–b binding proteins), Calvin cycle enzymes (ribulose phosphate, glyceraldehyde-3-phosphate, and ribose-5-phosphate), and starch biosynthesis enzymes (starch synthase and ADP-glucose pyrophosphorylase) was observed at 5% CO₂. These findings indicate coordinated regulation of multiple carbon metabolic pathways during cultivation under enriched CO₂. Overall, this study identifies key metabolites and regulatory targets involved in carbohydrate biosynthesis in H. rubescens, providing valuable insights for strain improvement and genetic engineering strategies in microalgal biotechnology.