Arrested Anaerobic Digestion Digestates Uncouple Denitratation from Denitritation During Shift from External to Intracellular Storage Carbon Sources

Introduction In recent years, partial denitrification anammox (PdNA) process has been developed in mainstream wastewater treatment as a low-carbon approach for biological nitrogen removal(Du et al., 2019). A prerequisite for effective PdNA process is the stable and reliable production of nitrite through partial denitrification (PdN) (Zhang et al., 2020), which is governed by the balance between denitratation (NO3- → NO2-) and denitritation (NO2- → N2). Volatile fatty acids (VFAs) have been widely exploited as effective electron donors to promote PdN and achieve nitrite accumulation. VFAs are commonly produced from organic waste through arrested anaerobic digestion (aAD) (Giduthuri & Ahring, 2023). Beyond serving as external substrates, VFAs can also be stored as intracellular storage carbon (ISC) polymers (Oehmen et al., 2007), primarily polyhydroxyalkanoates (PHA), with concurrent glycogen turnover. When external carbon is depleted, denitrification may continue by utilizing ISC (PHA or glycogen), potentially reshaping the competition between denitratation and denitritation. While partial denitrification under external carbon addition, especially VFAs, has been extensively studied, the competition between denitratation and denitritation during the ISC-driven phase remains poorly understood. Therefore, the objective of this study was to elucidate how denitratation and denitritation compete across the transition from external carbon-driven to ISC-driven denitrification with VFAs-containing aAD digestates as initial carbon sources. Two types of arrested anaerobic digestion (aAD) digestate, one was predominated by VFAs and the other containing a mixture of lactic acid and VFAs with a COD ratio of 1:0.62, were fed as carbon sources to activated sludge for denitrification dynamic studies. Meanwhile, as a widely used carbon source for PdN as well as the most common VFA component (Ji et al., 2025), pure acetate, was used as a positive control. Moreover, pure lactic acid was used in this experiment to provide counter evidence in support of VFA's role. Methodology Seed sludge was collected from a local municipal wastewater treatment plant. Before adding sludge to batch reactors, it was aerated for 1.5 h to deplete initial ISC. All batch tests were conducted at an initial COD/N ratio of 4, with a fixed initial COD of 50 mg/L. Buffer chemicals and trace elements were dosed identically across all groups before carbon and nitrate addition. Each reactor was purged with pure nitrogen gas for 2 min to establish anoxic conditions before experiment started. The initial mixed liquor volatile suspended solid (MLVSS) was controlled at 250.7 ± 15.2 mg/L. Samples were withdrawn hourly using sterile syringes and needles. Results Along with the denitrification timeline, the peak of nitrite accumulation synchronized with the peak of internal carbon storage until the 4th hour, both occurred when majority of the external carbon was utilized (Figure 1). Before external carbons were depleted, all four groups achieved substantial nitrite accumulation, achieving partial denitrification (PdN) efficiencies of 73.3±2.3%, 42.7±9.1%, 72.1±5.9% and 18.4±11.0% for VFA only digestate, VFAs + lactic acid digestate, pure acetate, and pure lactic acid, respectively. As shown in Figure 2, the concurrent PHA synthesis and phosphate release also observed along the denitratation suggest ISC formation driven by denitrifying polyphosphate-accumulating organisms (dPAOs). The corresponding specific PHA synthesis amounts were 110.8 ± 2.9, 52.5 ± 2.5, 108.3 ± 25.7, and 27.5 ± 2.4 mg PHA/g VSS, with phosphate release of 4.04 ± 0.94, 2.54 ± 0.47, 12.33 ± 3.92, and 1.68 ± 0.20 mg P/g VSS for the aforementioned four types of external carbon sources, respectively. Although there was no external carbon available after the 4th hour, the denitratation and denitritation continued but the rates of the former were only 78%, 86%, 81%, and 89% of the latter. These results suggested that ISC-driven denitrification prefers denitritation over denitratation. The fact that dPAOs lack the respiratory nitrate reductase enzyme (nar) and cannot effectively use nitrate as electron acceptor was regarded as the mechanism responsible for such an uncoupled rates between denitratation and denitritation when denitrifying glycogen-accumulating organisms (dGAOs) are presence (Rubio-Rincon et al., 2017). Thereby, this study determined the different competition performance between denitratation and denitritation under external carbon stage and ISC stage when VFAs and lactic acid are provided as initial carbon sources. Conclusions This study showed a clear shift in denitrification behavior when carbon transitioned from external VFAs to ISC (primarily PHA). During the external carbon phase (0-4 h), VFAs-rich aAD digestate and acetate achieved higher nitrite accumulation and PdN efficiency, while the presence of pure lactic acid impaired its PdN performance. After biodegradable COD was depleted, denitrification continued using ISC, but denitritation exceeded denitratation, indicating that ISC-driven denitrification favors nitrite consumption rather than nitrite production. These results suggest that maintaining stable nitrite supply for PdNA will require strategies to sustain external carbon availability or limit ISC-driven denitritation when utilizing VFA-containing aAD digestates.