Enhanced carotenoid accumulation in Chloroccocum humicola under controlled CO2 and light conditions

Carotenoids are potent antioxidants and high-value bioactive compounds that green microalgae can efficiently synthesize. This study aimed to enhance carotenoid production in Chlorococcum humicola TISTR 8551 using a two-stage cultivation strategy in a 10-L air-lift photobioreactor (ALPBR), separating biomass accumulation and stress induction phases. During the "green stage" (9 days), cells were grown in modified BG-11 medium (N:P ratio 31:1) under 3,500 Lux white LED light with varying CO₂ concentrations (1-3 % v/v) to maximize biomass yield. In the subsequent "red stage" (15 days), environmental stressors, including elevated salinity and intensified light exposure, were applied to stimulate carotenoid biosynthesis. The highest total carotenoid content (38.72 ± 1.04 mg/L, 0.313 ± 0.018 mg/g biomass) was observed under 3 % CO₂ supplementation, likely due to enhanced photosynthetic carbon fixation and improved precursor availability via glucose metabolism. An optimal white light intensity of 25,000 Lux produced 32.03 ± 1.52 mg/L carotenoids. Additionally, the combination of 100,000 Lux white light with 1,600 Lux blue light significantly increased β-carotene content (6.98 ± 0.28 % of total carotenoids), while 2,400 Lux blue light yielded the highest astaxanthin level (5.01 ± 0.18 % of total carotenoids). These results highlight the synergistic effects of CO₂ enrichment, spectral light modulation, and stage-specific stress application in promoting targeted carotenoid biosynthesis. This study offers practical insights for optimizing large-scale microalgal pigment production in controlled photobioreactor systems.