This research examined the feasibility of raw fermentate for mainstream partial denitrification-anammox (PdNA) in a pre-anoxic integrated fixed-film activated sludge (IFAS) process. Fermentate quality sampled from a full-scale facility was highly dynamic, with 360-940 mg VFA-COD/L and VFA/soluble COD ratios ranging from 24% to 48%. This study showed that PdNA selection could be achieved even when using low quality fermentate. Nitrate residual was identified as the main factor driving the PdN efficiency, while management of nitrate conversion rates was required to maximize overall PdNA rates. AnAOB limitation was never observed in the IFAS system. Overall, this study showed PdN efficiencies up to 38% and PdNA rates up to 1.2 ± 0.7 g TIN/m 2 /d with further potential for improvements. As a result of both PdNA and full denitrification, this concept showed the potential to save 48-89% methanol and decrease the carbon footprint of water resource recovery facilities (WRRF) by 9-15%. Practitioner points• Application of PdNA with variable quality fermentate is feasible when the nitrate residual concentration is increased to enhance PdN selection.• To maximize nitrogen removed through PdNA, nitrate conversion rates need enhancement through optimization of upstream aeration and PdN control setpoints.• The IFAS PdNA process was never anammox limited; success depended on the degree of PdN achieved to make nitrite available.• Application of PdNA with fermentate can yield 48-89% savings in methanol or other carbon compared with conventional nitrification and denitrification.• Integrating PdNA upstream from polishing aeration and anoxic zones guarantees that stringent limits can be met (<5 mg N/L).

This study successfully revealed the importance of probe reliability and sensitivity with ion sensitive electrode (ISE) probes on achieving high partial denitrification (PdN) efficiency; and decreasing carbon overdosing events that cause the decline of microbial populations and performance of PdNA. In a mainstream integrated hybrid granule–floc system, an average PdN efficiency of 76% was achieved with acetate as the carbon source. Thauera was identified as the dominant PdN species; its presence in the system was analogous to instrumentation reliability and PdN selection and was not a consequence of bioaugmentation. Up to 27–121 mg total inorganic nitrogen/L/d, an equivalent of 18–48% of the overall total inorganic nitrogen removed, was achieved through the PdNA pathway. Candidatus Brocadia was the main anoxic ammonium oxidizing bacteria species that was seeded from sidestream and enriched and retained in the mainstream system with observed growth rates of 0.04–0.13 day−1. Moreover, there was no direct negative impact of methanol's use for post‐polishing on anoxic ammonium oxidizing bacteria activity and growth. Practitioner Points Stress testing with ISE sensors revealed the importance of probe reliability and sensitivity on PdN selection and PdNA performance. Up to 121 mg TIN/L/d was achieved via PdNA in a mainstream suspended hybrid granule–floc partial denitrification–anammox (PdNA) system. Candidatus Brocadia was the dominant AnAOB species with observed growth rates of 0.04–0.13 day–1. There was no direct negative impact of methanol's use for post‐polishing on AnAOB activity and growth.