Iron plaques as terminal electron acceptors optimize clostridial fermentation and nitrogen fixation in rice...

Fermentative Clostridium species associated with rice roots can contribute substantially to biological nitrogen fixation (BNF) in anoxic paddy soils, yet whether their BNF is regulated by the redox chemistry of rhizosphere remains unclear. Here, we show that iron plaques on rice roots function as terminal electron acceptors that reprogram Clostridium fermentation and thereby enhance BNF. In nitrogen-fixation microcosms, Clostridium sensu stricto I was selectively enriched under plaque-associated Fe(III)-reducing conditions, coinciding with elevated nitrogen fixation. Metabolomic profiling coupled with metabolic flux analysis revealed that Fe(III) reduction redirects a portion of carbon and electron flow from low-energy-yield solventogenesis toward high-energy-yield acidogenesis. This shift increases cellular ATP generation and expands the reductant pool, thereby benefiting the energetic and reductant demands of nitrogenase. Integrated transcriptomic and metagenomic analyses further identified NosR, a flavin mononucleotide-binding protein that is upregulated during Fe(III) reduction and may facilitate electron delivery to plaque-associated Fe(III). Our findings establish a mechanism in which iron plaque reduction optimizes fermentation for BNF, providing fundamental insights into coupled Fe-N cycling in rice rhizospheres and suggesting potential strategies for sustainable nitrogen management in flooded agroecosystems.