Humic and Fulvic Acid Fractions Differentially Regulate Methane-Dependent Arsenate Reduction in Paddy Soils

Arsenic (As) contamination in paddy soils threatens global food security because microbial reduction of arsenate (As(V)) to mobile arsenite (As(III)) drives As mobilization. Methane (CH₄)-dependent As(V) reduction (M-AsR) is a key route coupling CH₄ cycling to As release, yet how structurally distinct soil organic matter (SOM) fractions regulate this pathway remains poorly understood. Here we show that an aromatic, quinone-rich humic acid fraction enhances electron transfer and promotes coupling of CH₄ oxidation to As(V) reduction, accelerating iron (Fe)-As mineral dissolution and increasing As(III) release by ∼1.5-fold. Accordingly, copy numbers of NC10-targeted pmoA, ANME-2d-targeted mcrA, and arrA increased by 116.6%, 126.5%, and ∼2.4-fold, respectively. In contrast, a carboxyl-rich fulvic fraction promoted acetate accumulation, thereby making CH₄-driven metabolism thermodynamically unfavorable. NC10-targeted pmoA and ANME-2d-targeted mcrA signals consequently decreased by 95.3% and 89.6%, respectively, and M-AsR was largely blocked, with the CH₄-driven As(III) component increasing by only 47.9%. Crucially, humic acid acts as an electron shuttle linking CH₄ oxidation and As(V) reduction, while fulvic acid disrupts this coupling process via acetate accumulation, highlighting the need for molecular-level SOM characterization to predict As risks in flooded soils.