Denitrification, Hydrolysis and Internal Store Carbon in Low Dissolved Oxygen Biological Nutrient Removal

Introduction

Dissolved oxygen (DO) is a key factor in the activated sludge process, influencing aeration costs, nitrogen/phosphorus removal, and N2O emissions. Research shows nitrifiers and denitrifiers can coexist and function in low DO zones, enabling 'simultaneous nitrification-denitrification' (SND). This highlights microbial adaptation to suboptimal nitrogen removal conditions. While progress has been made on nitrification and microbial selection at low DO (WRF Project No. 5083, 2025), low DO denitrification during SND remains underexplored. Understanding its mechanisms and influencing factors is crucial for optimizing low DO BNR systems, enhancing nitrogen removal, reducing costs, and improving carbon management.

Among the critical processes in BNR, denitrification (DN) -the biological conversion of nitrogen oxide (nitrate [NO3], nitrite [NO2-]) to nitrogen gas (N2)-plays a pivotal role in removing nitrogen from wastewater. Conventionally, DN is considered an anoxic process, occurring in environments with low or negligible DO concentrations. However, studies have challenged this paradigm by demonstrating the potential for DN under low DO conditions, opening new avenues for optimizing BNR systems using aeration controls.

The interplay between DN, DO conditions and electron donor are particularly relevant in energy-efficient wastewater treatment designs, which aim to reduce aeration energy without compromising treatment efficacy. This paper explores the dynamics of DN in low DO environments within BNR systems providing insights into hydrolysis and internally store carbon utilization. Key factors such as microbial community structure, substrate availability, removal pathways and reactor design are analyzed to understand their roles in enabling DN under such conditions. The findings aim to inform the development of optimized BNR strategies that leverage low DO DN during SND to improve efficiency, reduce energy consumption, and address the growing need for sustainable wastewater treatment solutions.

Materials and Methods
To evaluate the effects of oxygen on denitrification kinetics, microbial communities, and substrate availability, including hydrolysis, several full-scale and pilot-scale facilities operating under low DO conditions were analyzed. Long-term operational data from these facilities were compared with baseline information from a literature review. Mixed liquor samples were collected for laboratory-scale studies, and microbial ecology was examined using Quantitative PCR (qPCR) on DNA extracts. These findings enhanced understanding of the role of oxygen in DN within SND systems.

Results
The performance of full-scale low DO BNR plants was evaluated using one-year average data from facilities with DO levels ≤0.6 mg/L. These plants, with varied configurations, climates, and capacities, showed TIN removal from 65—95% with influent COD:N ratios of 7.5—13. SND contributed ~50% of TIN removal, depending on configuration. Complete nitrification (effluent NH4+ <0.5 mgN/L) was achieved. High selector F/M and rbCOD bleed-through enhanced DN under low DO conditions, as observed by profiling and lab tests not included in the abstract.

To study DO's effect on DN rates, mixed liquor from a low DO (<0.5 mg/L) plant performing SND was tested in batch reactors at varying DO concentrations (Figure 2). Results show maximum NUR under anoxic conditions, with rapid declines as DO rises. At 0.5 mg/L DO, NUR decreases by ~50%, and no DN occurs at 1.0 mg/L, likely due to fully aerobic flocs and oxidized carbon. The DN's oxygen half-saturation inhibition coefficient (Kio) averaged 0.19 ± 0.08 mg/L across facilities. This is an important process modeling parameter to simulate SND.

The role of soluble substrate (Ss) in DN is well-studied, but less is known about the role of the carbon dynamics on DN in low DO systems. Particulate substrate (Xs) biodegradation begins with hydrolysis, a critical step in DN during SND. In wastewater treatment, hydrolysis involves extracellular enzymes in microbial flocs converting Xs to Ss, enabling bacterial growth and carbon availability for denitrification. This process, slower than heterotrophic growth, is the rate-limiting step for organic compound degradation and DN in SND. DN rates were tested in two NUR batch tests under anoxic and low DO conditions using settled wastewater and acetate (Figures 3). In anoxic tests, rapid NO3 reduction with sCOD removal was followed by slower NO3 reduction attributed to hydrolysis, with specific sCOD removal of 4.8 mg sCOD/mgNO3. In low DO tests, three NUR slopes were observed: rapid NUR using sCOD, slower NUR using stored COD, and the slowest NUR via hydrolysis. The higher sCOD removal (e.g., 6.2 mg sCOD/mgNO3) was likely due to aerobic oxidation for cell growth. Process model predictions were fitted to the measured NUR and the hydrolysis rates were 2.9 d-1 (anoxic) and 3.8 d-1 (low DO).

Conclusions
SND has the potential to maximize TIN removal with influent carbon while reducing energy and chemical dependency. Denitrification in low DO systems is a critical step in SND with many factors in play. Understanding internal stored carbon and hydrolysis are key aspects of denitrification, especially in low DO systems. This study establishes important kinetic parameters including hydrolysis rate that can be used to simulate denitrification in low DO systems.