Investigation of light-regulated proliferation and division in Haematococcus pluvialis using O-PTIR and stable isotope probing

The molecular basis of carbon allocation to proteins and lipids in relation to light-exposure physiology remains poorly understood, particularly in the context of Haematococcus pluvialis growth and metabolism. Therefore, this study aims to elucidate the impact of varying light spectra on H. pluvialis carbon flux, thereby establishing a framework linking light perception with cell division. An integrative methodological approach was developed, combining stable isotope probing (using NaH 13 CO₃) and high-resolution Optical Photothermal Infrared (O-PTIR) microspectroscopy to trace 13 C incorporation into newly synthesized biomolecules under red, blue, and white light. The results showed that red light induced a pronounced spectral red shift (Δν = 8–10 cm −1 at 1665 cm −1 ) associated with amide I vibrations, indicating enhanced protein biosynthesis. Conversely, blue light elicited a significant decline in protein levels from 25.1 ± 0.5% to 21.3 ± 0.4%, whereas lipids rose sharply from 15.2 ± 0.3% to 17.9 ± 0.2%, revealing a “stress-induced” lipid storage phase. O-PTIR visualization showed lipids in membranes and droplets, with proteins uniformly distributed, reflecting their functional compartments. The mid-division stage showed peak biomolecular activity, with protein intensity (602 a.u.) nearly double that of pre-division cells. In conclusion, this study establishes O-PTIR with isotope tracing as a powerful tool for understanding carbon flow and molecular remodeling at the single-cell level, enabling better algal biomass and high-value metabolite production.