Coupling anaerobic-oxic and integrated fixed-film activated sludge processes for removal and transformation of refractory organic matter in livestock and poultry digestate

The treatment of livestock and poultry digestate is particularly challenging owing to the prevalence of recalcitrant organic matter (ROM) and its inherently low biodegradability. A sequential treatment train coupling the anaerobic-oxic (A/O) process with an integrated fixed-film activated sludge (IFAS) system was proposed and evaluated, hypothesizing that their complementary mechanisms would enhance ROM removal. By optimizing hydraulic retention time, aeration rate, and dilution ratio, the coupled system removed chemical oxygen demand (COD) by up to 60% at a removal rate of 1.0 kg COD/(m 3 ·d). Molecular-level investigation via fluorescence spectroscopy and Fourier transform-ion cyclotron resonance mass spectrometry revealed distinct yet complementary transformation roles that the A/O process primarily drove oxidative fragmentation of complex aromatics with increased molecular diversity. In contrast, the subsequent IFAS process focused on selective dealkylation and dehydrogenation, demonstrating superior conversion performance for saturated and reduced molecules while enhancing molecular complexity and aromaticity. 16S rRNA sequencing and correlation analyses linked these functional niches to specialized microbial consortia: Firmicutes and Proteobacteria dominated the A/O reactor, supporting oxidative catabolism, whereas Chloroflexota , Bacteroidota , and Planctomycetota prevailed in the IFAS biofilm, associating with reductive transformations. This work elucidates a synergistic oxidation-reduction sequence enabled by coupling A/O and IFAS, providing an effective strategy for mitigating refractory agricultural wastewater. The findings offer fundamental insights into designing microbiome-driven, multi-stage biological treatment systems. • An A/O-IFAS system was developed for treating livestock digestate for COD removal at a high rate of 1.0 kg COD/(m³·d). • Revealed complementary molecular mechanisms: A/O drove oxidative fragmentation while IFAS reduced transformations. • FT-ICR MS revealed molecular shifts across A/O and IFAS phases. • Functional microbial niche partitioning of Firmicutes / Proteobacteria vs. Chloroflexota / Bacteroidota was identified.