Magnetite-assisted enhancement of stability and resilience in anaerobic digesters against hydraulic overloading and volatile fatty acid shocks.

Anaerobic digestion (AD) stability is often compromised under operational disturbances due to imbalances between acidogenesis and methanogenesis, where insufficient interspecies electron transfer (IET) can limit syntrophic oxidation of volatile fatty acids (VFAs). Conductive additives are widely recognized to enhance direct IET (DIET), yet the responses of electroactive microbial communities that support process resilience to operational disturbances remain unclear. Here, duplicate continuous AD reactors with and without submicron magnetite (Fe3O4) supplementation were operated under sequential VFA shocks and stepwise hydraulic retention time (HRT) reductions to examine process performance, sludge activity, and RNA-level microbial community dynamics. Following the second VFA shock of 4.5 g chemical oxygen demand (COD)/L, magnetite-amended reactors stabilized within three days, whereas controls required up to ten days, reflecting improved syntrophic VFA oxidation, especially of propionate. Under stepwise HRT reduction, magnetite-amended reactors sustained a methane yield of 0.19 L/g COD fed even at a 10-d HRT, whereas controls yielded only 0.01 L/g COD fed with VFA accumulation. Magnetite also reduced toxic H2S accumulation with concurrent S0 formation, suggesting an additional benefit for sulfide control. The improved performance coincided with higher electron transport and specific methanogenic activities, greater retention of putative electroactive microorganisms, and persistent dominance of Methanothrix. Potential bacterial partners of Methanothrix shifted with disturbance conditions, with Anaerolineaceae and Syntrophus retained during VFA shocks and Tolumonas, Syntrophus, and Geobacter becoming more prominent under short-HRT conditions. These findings suggest that magnetite contributes to AD resilience, potentially through electro-syntrophic interactions that support process stability during operational disturbances.