This work presents the direct crude glycerol use by high cell-density fermentation of Escherichia coli expressing recombinant glycoside hydrolase that cleaves steryl glucosides, one of the main biodiesel contaminants. Optimized fermentation with crude glycerol reached the biomass concentration of 30 g/l with the 0.285 U/mgDCW specific activity, which is comparable with the pure glycerol production. The expression of the inclusion bodies was eliminated by the increase of the induction temperature to 40 °C. The produced enzyme was able to hydrolyze 300 ppm of steryl glucosides, the major biodiesel contaminants, and reached 100 % conversion. This model approach of waste carbon recycling (crude glycerol) for biorefinery-beneficial recombinant enzyme production is in line with the sustainable circular bioeconomy concept.