Eosin Y enhances bisulfite-driven H2 photoproduction in Chlorella pyrenoidosa by promoting carotenoid-associated photoprotection and preserving PSII activity.

Bisulfite enhances algal H2 photoproduction by scavenging oxygen and establishing hypoxic conditions favorable for hydrogenase (H2ase) activity. However, bisulfite treatment can also increase oxidative stress, impair photosystem II (PSII), and restrict photosynthetic electron supply for H2 production. Here, NaHSO3 treatment of Chlorella pyrenoidosa established hypoxia and increased H2 production approximately fivefold but substantially impaired PSII activity. Co-application of eosin Y alleviated PSII impairment and increased H2 production approximately 30-fold relative to the untreated control and sixfold relative to NaHSO3 treatment alone. Under illumination, eosin Y induced a modest 1O2-associated fluorescence response that was accompanied by increased carotenoid accumulation without detectable impairment of PSII activity. Under NaHSO3-induced hypoxic conditions, eosin Y further promoted carotenoid accumulation and remodeled carotenoid biosynthesis, including increased expression of the lycopene ε-cyclase gene, decreased expression of the cytochrome P450-type carotene β-ring hydroxylase gene, and enrichment of α-carotene. These responses were associated with lower intracellular •OH-associated fluorescence and improved PSII activity under bisulfite-induced hypoxic conditions. Inhibition of carotenoid biosynthesis markedly attenuated the PSII-protective effect of eosin Y, supporting a contribution of carotenoid accumulation to PSII protection. Consistent with the improved PSII function, eosin Y increased PSII electron transport without further altering dissolved oxygen or in vitro H2ase activity under NaHSO3 treatment. Thus, eosin Y-associated carotenoid accumulation and remodeling contributed to alleviating the oxidative limitation imposed by NaHSO3 and helped preserve PSII-dependent electron supply for H2 photoproduction. These findings demonstrate a potential strategy for coordinating NaHSO3-induced hypoxia with endogenous carotenoid-associated photoprotection to improve light-driven H2 production in microalgae.