Pichia pastoris fermentation for phytase production using crude glycerol from biodiesel production as the sole carbon source

Abstract

Abstract Efficient utilization of crude glycerol, a by-product from biodiesel production, could bring significant economic and environmental benefits. In this work, a low-grade glycerol was used as the sole carbon source in phytase production with recombinant Pichia pastoris possessing a pGAP-based constitutive expression vector. In batch and fed-batch modes, the effects of important cultivation conditions were investigated using both analytical and biodiesel glycerols in batch and fed-batch fermentations. The following factors were analyzed: initial glycerol concentration, dissolved oxygen level, and the effect of feeding strategy on cell growth biomass and protein production. Significant cell growth inhibition was observed in batch fermentation when the initial crude glycerol concentration was as high as 70 g/L. However, such inhibition was overcome in fed-batch mode by starting the cultivation with a lower crude glycerol level. Finally, cell densities and phytase activity levels of as high as 146 g (dry cell weight)/L broth and 1125 U/mL supernatant were achieved in the fed-batch fermentation with crude biodiesel glycerol as the sole carbon source. The study has proven the potential of using crude glycerol from biodiesel production as the carbon source for industrial scale phytase mass production in high cell density fermentations with recombinant P. pastoris.