<p>The role of organic carbon source as electron donor in incomplete denitrification, particularly in nitrite (NO₂⁻) accumulation, remains crucial yet poorly understood. A detailed understanding of carbon and nitrogen metabolic interactions is essential for advancing technologies that integrate partial denitrification (PD) with anammox. In this study, the carbon transformation and gradient utilization of various volatile fatty acids (VFAs) were explored to elucidate their impacts on nitrate (NO₃⁻) and NO₂⁻ reduction during PD. Long-term experiments revealed that composite VFAs (a mixture of acetate, propionate and butyrate) achieved the highest nitrate-to-nitrite transformation ratio (NTR) of 79.1%, outperforming single VFA (69.5% with acetate and 69.4% with propionate). The NO₂⁻ accumulation during PD was strongly influenced by the utilization of exogenous, endogenous and extracellular carbon, which varied significantly with VFAs type and dosage. Polyhydroxybutyrate (PHB) served as the primary endogenous electron donor in acetate-driven PD, promoting modest NO₂⁻ accumulation, while polyhydroxyvalerate (PHV) along with glycogen (Gly) was the key contributor in propionate-driven PD, supporting complete NO₃⁻ reduction. In contrast to single VFA-driven PD, the lower levels and delayed utilization of PHB and PHV in composite VFAs-driven PD enabled more stable and efficient NO₂⁻ accumulation. Furthermore, metagenomic analysis illuminated that the transition from single VFA to composite VFAs strengthened the potential for both electron production and their transport to NO₃⁻ reductase. <i>Thauera</i> was always the core denitrifier demonstrating strong adaptability to various VFAs. This study provides mechanistic insights into organic carbon-regulated NO₂⁻ accumulation, filling the gap regarding dynamic changes in carbon utilization during PD.</p>