Nanomagnetite Promotes Sub-Thermophilic Anaerobic Digestion by Enhancing Electron Transfer and Electron Bifurcation

Abstract

Subthermophilic anaerobic digestion accelerates hydrolytic acidification to boost methane production, yet this temperature range can suppress methanogenic activity, thereby leading to a kinetic mismatch between rapid substrate influx and limited methanogenic conversion. In this study, nanomagnetite was employed to mitigate this inhibition by enhancing direct interspecies electron transfer (DIET) and electron bifurcation (EB). Results showed that the subthermophilic (45 °C) group achieved 13.3% higher cumulative methane production compared to the mesophilic control (37 °C), with nanomagnetite supplementation yielding an additional 11.4% increase. Mechanistically, subthermophilic digestion generated high levels of ethanol, which was subsequently oxidized to release more energy to drive electron efflux and reduce CO₂ to methane. However, the electron flow was slowed by the low metabolic activity of methanogens under subthermophilic conditions. Nanomagnetite facilitated extracellular electron transfer and enhanced EB to redirect the impeded electron flow to drive ATP synthesis. Consequently, the 37.1% increase in ATP production in the 45 °C-NM group likely supported F₄₃₀ biosynthesis and restored the McrA activity under subthermophilic conditions. These findings demonstrated that nanomagnetite could enhance DIET coupling with EB to alleviate the inhibition on methanogens caused by subthermophilic conditions and achieve efficient methanogenesis.