Conductive Fe₃O₄ drives a stress-dependent shift in electron-transfer strategies during anaerobic tetramethylammonium hydroxide degradation.

Anaerobic treatment of tetramethylammonium hydroxide (TMAH) wastewater is often constrained by metabolic disruption under high TMAH loading. Conductive Fe₃O₄ has shown promise for enhancing anaerobic bioprocesses; however, the dominant mechanism governing its enhancement under different metabolic conditions remains unclear. Here, anaerobic reactors were operated under stepwise increases in TMAH concentration (1-10 g/L) to compare the effects of soluble Fe²⁺ and conductive Fe₃O₄ using reactor performance, electrochemical characterization, intracellular reducing-equivalent measurements, and integrated metagenomics and metatranscriptomics analyses. At low-to-moderate TMAH loadings (1-5 g/L), Fe₃O₄ and Fe²⁺ exhibited comparable improvements in TMAH degradation and methane production, together with elevated soluble Fe²⁺ and reduced ferredoxin, indicating that dissolved iron was the dominant contributor. Under severe TMAH stress (10 g/L), both iron amendments improved reactor performance, whereas Fe₃O₄ became markedly more effective than Fe²⁺, achieving complete TMAH removal and over twofold higher methane production than the unamended control. Electrochemical analyses showed that Fe₃O₄ enhanced extracellular electron-transfer activity, accompanied by intensified riboflavin-like redox signals. Integrated metagenomic and metatranscriptomic analyses further revealed concurrent enrichment of electroactive bacteria and syntrophic methanogens, together with increased transcription of genes involved in intracellular electron metabolism, particularly those associated with F₄₂₀-dependent C₁ metabolism and HdrABC-MvhADG-mediated electron bifurcation. Collectively, these findings unravel a stress-dependent transition in the mechanism underlying Fe₃O₄ enhancement, from dissolved-iron-supported intracellular reducing-equivalent metabolism under low-to-moderate TMAH loadings to conductivity-associated coordination between extracellular electron transfer and intracellular methanogenic metabolism under severe TMAH stress. This mechanistic insight provides a basis for optimizing conductive iron mineral-assisted anaerobic treatment of high-strength TMAH wastewater.