Humic acid induced astaxanthin accumulation by changing GABA, polyamines, and H2O2 levels in Haematococcus lacustris at the stationary growth phase.

Abstract

Humic acid (HA) is a redox-active organic compound that can regulate cell metabolism to produce antioxidant metabolites against oxidative stress. Haematococcus lacustris is a green microalga and is found to be a rich source of astaxanthin. In this research, the impact of HA was studied on the growth mechanisms and production of antioxidant metabolites through dynamic responses of pigments, proteins, carbohydrates, secondary messengers of H₂O₂ and Ca²⁺, gamma-aminobutyric acid (GABA), and enzyme activities in H. lacustris. Results revealed that HA at 80 µM concentration is a suitable treatment to induce astaxanthin production and cell growth. Cell numbers increased significantly under HA80, and the trend was to enter the red aplanospore phase at the stationary growth phase. High HA concentration (120 µM) increased astaxanthin content but considerably reduced cell number and size. HA80 enhanced astaxanthin (5.39 mg L^-1), flavonoid (15.64 mg g^-1 DW), and phenolic (55.64 mg g^-1 DW) contents after 9 days of induction time, which was accompanied by a significant reduction in the chlorophyll pigments, proteins, and carbohydrate contents. The increase in total phenolic content was associated with enhanced phenylalanine ammonia-lyase activity. H₂O₂ accumulation decreased by HA80 at the late stationary growth phase. Putrescine and spermidine contents were promoted under HA80, while gamma-aminobutyric acid (GABA) and Ca²⁺ contents were reduced from the logarithmic phase to the early stationary growth phase. Succinate dehydrogenase (SDH) activity was promoted in the TCA cycle, and the GABA shunt was activated to regulate the ROS level. Findings indicate that the impact of HA on cell growth and astaxanthin production is associated with HA concentration and cell growth phase. HA can regulate ROS levels at the stationary growth phase by inducing polyamine metabolism and an antioxidant defense system.