Unveiling the Coupling Mechanism between Central Carbon and Nitrogen Metabolism of <i>Pseudomonas stutzeri</i>: Polyhydroxyalkanoate-Enhanced Electron Supply for Denitrification

High nitrogen levels in drinking water sources pose a public health threat and have drawn significant attention. In situ high-efficiency ecological restoration technology using polyhydroxyalkanoates (PHAs) can effectively enrich target microorganisms and enhance denitrification in water bodies. However, the mechanisms behind PHA-driven carbon metabolism, nitrogen transformation, and electron transfer in denitrification remain unclear. This study investigated Pseudomonas stutzeri using sodium acetate (NaAc) or sodium 3-hydroxybutyrate as the sole carbon source. The PHA group achieved superior nitrogen removal (171.44 mg/L N, 13.39 mg N/mg DNA) compared to the NaAc group (83.83 mg/L N, 7.98 mg N/mg DNA), driven by elevated NADH levels and an increased NADH/NAD+ ratio, facilitating electron transfer to denitrifying enzymes (i.e., Nap, Nir). The metabolic flux shifted, with the tricarboxylic acid (TCA) cycle decreasing 7.08-fold and the pentose phosphate pathway (PPP) increasing 2.53-fold, optimizing reducing equivalent production for nitrogen assimilation and denitrification. A potential mechanism for PHA-enhanced denitrification and new insights into how carbon substrates regulate microbial functions in denitrification were then proposed.