High-Protein Enhancement of Spirulina via CO ₂ Nanobubble Cultivation and Post-Harvest Mechanical Milling: Biochemical and Environmental Insights

In recent years, Spirulina platensis has gained attention as a sustainable microalga for protein, pigment, lipid, and bioactive compound production. This study integrates upstream and downstream optimization through (i) pre-cultivation CO2 nanobubbles (CO2-NBs) to enhance inorganic carbon delivery and (ii) post-harvest mechanical milling for biomass refinement. Biochemical integrity was evaluated using Raman spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, and proximate analysis. Meanwhile, CO2-NBs characteristics, including size and zeta potential, were assessed in an aquadest medium. Pre-cultivation with CO2-NBs significantly enhanced protein content from 5.21 to 32.3% on a dry-weight basis following 30 min of injection. This improvement was supported by the intensified Raman shift around 1005 cm−1, associated with phenylalanine, alongside FTIR peaks near 1650 and 1050 cm−1, confirmed amide I and II as protein indicators. Post-mechanical milling of CO2-NBs with 30 min of injection time effectively reduced dry cell weight (DCW) S. platensis size from 1009 to 395−566.2 nm. Life cycle assessment (LCA) limited to cultivation highlighted the environmental advantage of CO2-NBs, reducing emissions to 473.9 kg CO2e/kg dry weight relative to 713.1 kg CO2e/kg in the control.