Polyhydroxyalkanoates (PHA) are a class of biodegradable polymers with physicochemical properties similar to
conventional plastics. However, their large-scale commercial application has been hindered by high production costs. This study utilized waste glycerol as a low-cost carbon source, combined with the activated sludge process, to synthesize PHA. Through aerobic cultivation with alternating feast/famine alternation strategies and batch experiments, this study systematically investigated the effects on the synthesis of PHA, as well as the production of related by-products such as extracellular polymeric substances (EPS) and soluble microbial products (SMP), and explored the associated carbon source competition mechanisms. The experimental results demonstrated that: (1) Activated sludge can efficiently synthesize PHA from glycerol, with both PHA yield (YPHA/S) and maximum PHA accumulation (PHAm) showing a significant positive correlation with glycerol concentration. The highest YPHA/S (0.95 g PHA/g COD) and maximum PHAm (7.23%) were achieved at a glycerol concentration of 600 mg COD/L; (2) EPS, like PHA, is a polymer synthesized by microorganisms using external carbon sources, and there is a significant competitive relationship between EPS and PHA for carbon utilization; (3) SMP, as a by-product of PHA synthesis in activated sludge, exhibited a similar trend to EPS and was negatively correlated with PHA production. This study provides a theoretical foundation and technical support for the large-scale production of PHA using waste glycerol, offering a cost-effective approach while enabling the resource utilization of waste glycerol, with both economic and environmental benefits.