Boosting biomass and superoxide dismutase yield in Tetraselmis chuii via two-stage cultivation and adaptive...

Microalgae, such as Tetraselmis chuii, are potent sources of natural antioxidants, attributed to their adaptation to oxidative stress in harsh marine environments. This study aimed to investigate superoxide dismutase (SOD) production in T. chuii, a microalga recently approved as a novel food by the European Union. Although oxidative stress application was hypothesized to enhance SOD biosynthesis, direct stress exposure potentially compromises biomass accumulation, creating a trade-off between cell density and antioxidant production. Therefore, a two-stage cultivation strategy was employed: the first stage focused on optimizing biomass through medium enhancement until an adequate cell density was achieved in the stationary phase, followed by the controlled induction of oxidative stress to stimulate SOD overproduction without significantly impacting the established biomass. F medium, supplemented with NH₄Cl, was selected as the basal cultivation medium. Key components, such as NH₄Cl, NaH₂PO₄, CuSO₄, and MnCl_2, were optimized using Response Surface Methodology (RSM), increasing biomass from 416.67 mg/L to 564.44 mg/L and SOD activity from 1479.23 U/g to 2757.27 U/g. Various stressors, including trace heavy metals, oxidants, and salinity modulation, were applied to elevate SOD production further. CuSO₄, used as a stressor, proved most effective at 5.0 mg/L, increasing SOD activity to 5774.76 U/g compared to 2307.62 U/g in the control. To build microalgal tolerance towards copper-induced stress, an Adaptive laboratory evolution (ALE) experiment was conducted over 27 weeks, resulting in a post-ALE strain with a 61% increase in SOD activity. Overall, this study demonstrates the effectiveness of medium optimization, stress induction, and ALE in augmenting antioxidant production in microalgae.