Squalene production from biodiesel‐derived crude glycerol as the substrate by <i>Pichia pastoris</i> in batch and fed‐batch systems

Abstract This study reports the production of squalene and other value‐added metabolites from crude glycerol by the yeast Pichia pastoris . To the best of our knowledge, this is the first study to demonstrate the utilization of crude glycerol for squalene biosynthesis in P. pastoris , with process intensification achieved through fed‐batch cultivation in a bioreactor. A maximum squalene titer of 70 mg L −1 was achieved using 1% (v/v) crude glycerol along with 0.2 mg L −1 added terbinafine in mineral salt media. Various biokinetic models, modified‐Gompertz, Luedeking‐Piret, Aiba, and Andrews models, were applied to understand the kinetics of squalene accumulation by P. pastoris . The Modified‐Gompertz model accurately predicted the maximum squalene production, whereas the Aiba and Andrews models described the inhibitory effect caused by crude glycerol. Scale‐up experiments performed using a laboratory‐scale stirred tank bioreactor demonstrated enhanced squalene accumulation by 1.67‐fold under batch operation (117 mg L −1 ) and by up to 2.94‐fold under fed‐batch operation (206 mg L −1 ), relative to the shake flask cultivation study. High‐resolution mass spectrometry analysis of the spent media revealed that, in addition to squalene accumulation by the yeast, arabitol, succinic acid, mannitol, benzoic acid, and citric acid were produced as extracellular products from crude glycerol.