Metabolomics-based approach to explore growth phase-dependent markers in cultured diatom Chaetoceros tenuissimus

Chaetoceros tenuissimus is a cosmopolitan marine diatom whose metabolism has been little investigated. In this study, we examined the alterations of the metabolite profile between different growth phases (exponential and stationary phase) in cultured C. tenuissimus and identified growth phase-dependent candidate marker metabolite. First, the preparation methodology was optimized, focusing on extraction solvent. Metabolites of the cultured diatom (exponential phase 2.4 × 10⁶ cells/mL, stationary phase 3.1 × 10⁶ cells/mL) were extracted using two solvents with different constituents (solvent 1, methanol:H₂O:chloroform = 5:2:2; solvent 2, methanol:ethanol:chloroform = 1:3:1). Gas chromatography mass spectrometry (GC/MS)-based metabolomics successfully detected 43 water-soluble metabolites in both solvents. The metabolic features were dependent on the growth phase: amino acid levels were higher in the exponential phase, whereas sugars and alcohols were more abundant in the stationary phase. Solvent 1 was superior in the recovery of the candidate metabolite that had a retention time of 18.13 min and predominantly contributed to discrimination between the growth phases; the metabolite level was higher in the stationary phase than in the exponential phase. The candidate metabolite was identified as mannonic acid by using GC/MS and liquid chromatography tandem mass spectrometry. Higher levels of mannonic acid during the stationary phase were also observed in other three diatom species. This study provides further insight into the use of metabolomics in the evaluation of physiological conditions of diatoms and suggests that mannonic acid content is a potential biomarker of the growth phase in cultured diatom cells.