Carbon availability dictates the stability of nitrate-vanadium co-remediation in stratified biofilters.

Thermodynamic hierarchies constrain the bioremediation of groundwater co-contaminated with nitrate (NO3-) and pentavalent vanadium (V(V)), denitrification preferentially consumes electron donors that would otherwise support metal reduction. Here, we show that spatial stratification of lignocellulosic residues (wheat straw → corn straw → corncob) can transiently alleviate competition between these competing processes, although system performance remains ultimately governed by carbon availability and kinetics. Over 330 days of operation, the stratified biofilter exhibited a biphasic response: (i) a carbon-sufficient phase (0 - 88 d) that enabled synergistic co-removal, increasing NO3- and V(V) loading capacities by up to 6.3-fold and 4.0-fold, respectively, relative to single-substrate controls; and (ii) a carbon-limited phase (88 - 330 d) in which denitrification persisted (>50% removal) while V(V) reduction collapsed (≈0%). Spatially resolved metagenomics (n = 15) revealed the mechanism as a thermodynamic "metabolic triage": under carbon limitation, microbial communities maintained denitrification pathways but selectively down-regulated V-reduction modules (sulfite reductase and multiheme cytochromes) by 59% - 69%. While distinct functional niches emerged-characterized by rapid efflux (top), deep reduction (middle), and sequestration (bottom), spatial organization alone could not override thermodynamic limits. Our findings establish that sustained metal co-remediation requires dynamic carbon management strategies to actuate latent genetic potential, providing a design framework for overcoming competitive inhibition in engineered aquifers.