Carbon Dioxide Fixation Cascade for the Production of Single-Cell Protein by <i>Clostridium ljungdahlii</i> and <i>Yarrowia lipolytica</i>

<i>Clostridium</i> species and yeast single-cell protein (SCP), produced via industrial fermentation, offer a sustainable, high-protein alternative to conventional sources while advancing circular economy goals through CO₂ valorization. Here, we developed an integrated two-stage bioprocess for SCP production from CO₂. First, <i>Clostridium ljungdahlii</i> converted CO₂ and H₂ into SCP and acetate via gas fermentation. Optimizing nitrogen and sulfur feeding based on acetate synthesis rate increased acetate titer from 6 to 53.2 g/L and biomass from 1.1 to 2.8 (OD₆₀₀). The fermentation broth was then directly used by <i>Yarrowia lipolytica</i>. Without pH control, dry cell weight (DCW) reached 4.7 g/L (67.0% protein, 6.5% lipid). With H₂SO₄-maintained pH 7, DCW reached 17.6 g/L (75.0% protein, 11.1% lipid). Notably, pH control using acetate yielded the highest DCW of 74.6 g/L (57.0% protein, 10.5% lipid). It should be noted that the highest biomass titer was achieved only when 130 g/L external acetate was added with pH control. This work demonstrates an efficient strategy for converting CO₂ into valuable microbial protein, advancing scalable SCP production.