Identification and Mitigation of Inhibitory Substances Contained in High-Salinity Crude Glycerol Generated from Biodiesel Production for Polyhydroxyalkanoate Synthesis by <i>Haloferax mediterranei</i>

High-salinity crude glycerol generated from biodiesel production poses significant challenges to microbial valorization due to inhibitory ingredients that severely limit microbial growth. This study identified and mitigated inhibitory substances contained in high-salinity glycerol sludge to enable its conversion to polyhydroxyalkanoates (PHAs) by the extreme halophilic archaeon <i>Haloferax mediterranei</i>. The long-chain fatty acids (LCFAs) were consistently identified as the primary inhibitors by liquid chromatography-mass spectrometry, Fourier transform infrared spectroscopy, and ultraviolet-visible spectroscopy. Acid precipitation at pH 2 efficiently removed these LCFAs, substantially reducing the required feedstock dilution from 23 to 3 times, improving PHA titer by 40%. Furthermore, this dilution reduction also increased the feedstock salinity utilization, achieving a 46% reduction in external salt supplementation for <i>H. mediterranei</i> growth. In contrast, overliming and arrested anaerobic digestion were confirmed to be ineffective in inhibitor removal. This study provides deep insights into inhibitor chemistry and presents acid precipitation as an effective pretreatment strategy for waste valorization of high-salinity crude glycerol.