Extending the green stage of Haematococcus pluvialis as a crucial precursor for efficient astaxanthin production: Optimization via Taguchi design

The microalga Haematococcus pluvialis can accumulate the natural antioxidant astaxanthin up to 5 % of its dry weight under multi-stress conditions. Increasing this percentage remains challenging; therefore, the key to efficient astaxanthin production lies significantly on the formation of a vigorous, high-density, and synchronized green-cell population prior to the induction of carotogenesis. Despite its importance, optimization of the vegetative growth phase is often underestimated. This study employs an L9-Taguchi design of experiments to systematically investigate the combinatorial effects of four key cultivation factors - temperature, sodium bicarbonate, nitrogen, and phosphorus - on the vegetative growth of H. pluvialis. Temperature emerges as the most influential factor for biomass production, showing an adverse effect with its increment. Nitrogen and phosphorus limitation combined with sodium bicarbonate supplementation induces a premature shift to the non-proliferative red stage. Overall, the optimal conditions identified for vegetative stage maintenance and biomass maximization are: 22 °C, 250 mg L⁻¹ sodium bicarbonate, 150 mg L⁻¹ nitrogen and 40 mg L⁻¹ phosphorus. These conditions result in the production of 1.53 g L⁻¹ green dry biomass after 15 days of cultivation, outperforming by 0.3 g L⁻¹ suboptimal green phase conditions. Post-verification of the Taguchi-derived predictions is experimentally demonstrated paving the way for the formulation of dense and robust green-cell populations that will be concomitantly transferred to the red stage.