BackgroundDiatoms have been viewed as ideal cell factories for production of some high-value bioactive metabolites, such as fucoxanthin, but their applications are restrained by limited biomass yield. Mixotrophy, by using both CO2and organic carbon source, is believed effective to crack the bottleneck of biomass accumulation and achieve a sustainable bioproduct supply.ResultsGlycerol, among tested carbon sources, was proved as the sole that could significantly promote growth ofCylindrothecasp. with illumination, a so-called growth pattern, mixotrophy. Biomass and fucoxanthin yields ofCylindrothecasp., grown in medium with glycerol (2 g L−1), was increased by 52% and 29%, respectively, as compared to the autotrophic culture (control) without compromise in photosynthetic performance. AsCylindrothecasp. was unable to use glycerol without light, a time-series transcriptomic analysis was carried out to elucidate the light regulation on glycerol utilization. Among the genes participating in glycerol utilization,GPDH1,TIM1andGAPDH1, showed the highest dependence on light. Their expressions decreased dramatically when the alga was transferred from light into darkness. Despite the reduced glycerol uptake in the dark, expressions of genes associating with pyrimidine metabolism and DNA replication were upregulated whenCylindrothecasp. was cultured mixotrophically. Comparative transcriptomic and metabolomic analyses revealed amino acids and aminoacyl-tRNA metabolisms were enhanced at different timepoints of diurnal cycles in mixotrophicCylindrothecasp., as compared to the control.ConclusionsConclusively, this study not only provides an alternative for large-scale cultivation ofCylindrotheca, but also pinpoints the limiting enzymes subject to further metabolic manipulation. Most importantly, the novel insights in this study should aid to understand the mechanism of biomass promotion in mixotrophicCylindrothecasp.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02338-8.