Inhibition and Recovery of Anammox Bacteria on Biofilm Carriers Exposed to Free Ammonia and Free Nitrous Acid

Abstract

Anaerobic ammonium oxidation (anammox) is an energy-efficient biological process for nitrogen removal that has been widely implemented in sidestream systems, expanded into mainstream applications, and increasingly applied to high-strength industrial waste streams. Despite its advantages, anammox performance is sensitive to inhibition by free ammonia (FA) and free nitrous acid (FNA). While inhibitory thresholds for these species are well documented, less is understood about the reversibility of inhibition and the extent to which recovery depends on the pathway by which inhibitory conditions are achieved, defined here as whether inhibition results from changes in pH or changes in total nitrogen concentration. This study evaluated anammox inhibition and recovery under controlled FA and FNA exposures using bench-scale moving bed biofilm reactors (MBBRs) with carriers with mature biofilm from a sidestream PNA process. Biofilm carriers were subjected to approximately 20-hour exposure periods across a range of ammonia and nitrite concentrations, with pH-controlled conditions used to isolate ammonia and nitrite speciation, enabling independent evaluation of FA and FNA formation. Maximum anammox activity tests (MAATs) were conducted before, during, and after exposure to quantify percent inhibition, recovery potential, and time to maximum recovery. Statistical analyses compared FA and FNA concentrations with total species metrics, including total ammonia nitrogen (TAN) and total nitrite species (TNS). Results demonstrated fundamentally different inhibition and recovery dynamics for FA and FNA. FNA caused rapid and near-complete inhibition (>85%) at low concentrations (<0.04 mg N/L), yet recovery was consistently high (>85–100%) following removal of the inhibitory condition. In contrast, FA produced more variable inhibition but resulted in reduced recovery potential and longer recovery times at elevated concentrations. Notably, TAN exhibited stronger relationships with both inhibition (adjusted R² = 0.639, p = 0.0117) and recovery behavior than FA alone, indicating that ammonia-related inhibition is not fully described by NH₃ concentration in isolation. Recovery time increased significantly with FA concentration (adjusted R² = 0.509, p = 0.0345), whereas no meaningful relationship was observed for FNA. These findings demonstrate that FNA acts as a rapid but largely reversible inhibitor, while FA exposure leads to more persistent inhibition with reduced recoverability. The results further show that anammox response depends not only on inhibitor concentration, but also on how those conditions are established through interactions between pH and bulk nitrogen. This distinction is particularly relevant for sidestream and high-strength treatment systems, where controlling both nitrogen loading and pH is critical for maintaining stable and resilient anammox performance.