Physiological responses and metabolic mechanisms of marine Chlorella under hypotonic stress: Implications for nutrient removal and starch accumulation

Tidal estuarine systems are characterized by significant salinity fluctuations and are highly susceptible to nutrient pollution. Chlorella vulgaris, a marine microalga, shows great potential for nutrient assimilation and biomass production, with a broad tolerance to salinity, making it a promising candidate for ecological restoration in such environments. However, its physiological and metabolic responses to hypotonic stress in tidal estuaries remain unclear. This study simulated tidal estuarine conditions using three salinity gradients (5 ‰, 15 ‰, and 25 ‰) and assessed nutrient removal efficiency and starch accumulation by measuring nitrogen, phosphorus, and starch content, while transcriptomic analysis was used to reveal changes in physiological responses and metabolic pathways. Pigments content and malondialdehyde (MDA) content account for the photosynthesis and level of the hypotonic stress. Differential expression was analyzed to identify key metabolic pathways involved. The results showed Chlorella under hypotonic stress exhibited enhanced nutrient assimilation and starch accumulation, chlorophyll a increased continuously at the expense of chlorophyll b and MDA accumulation primarily occurred during the first three days of cultivation while SOD activity on the fifth day is ranked from high to low: 5 ‰, 15 ‰, 25 ‰ and Control. Antioxidant pathways, photosynthesis, glycolysis, and nutrient assimilation appeared to be closely linked in energy metabolism. Differentially expressed genes analysis revealed downregulation of many biological processes consuming ATP and NADPH, while nitrogen assimilation (via glutamate synthesis) and phosphate transmembrane transport were significantly upregulated, alongside the enhancement of antioxidant pathways and photosynthesis. Overall, these findings demonstrate the substantial advantages of Chlorella in nutrient removal and starch accumulation under hypotonic stress, offering valuable insights for its application in ecological restoration in tidal estuaries.