Carbon Dioxide Fixation Cascade for the Production of Single-Cell Protein by Clostridium ljungdahlii and Yarrowia lipolytica.

Clostridium 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, Clostridium ljungdahlii 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 Yarrowia lipolytica. 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.