Synergistic enhancement of microbial denitrification by coupled nanoscale zero-valent iron and graphite powder: Mechanisms of electron flux optimization, iron bioavailability, and metabolic network coordination

Nanoscale zero-valent iron (nZVI) has shown promise in enhancing microbial denitrification for low carbon-to‑nitrogen (C/N) ratio wastewater treatment. However, its high reactivity leads to unstable performance and inhibitory effects on microbial metabolism. While nZVI modifications have been widely explored, the regulatory potential of co-existing inert materials in mediating nZVI-microbe interactions remains unaddressed. This study introduced a coupled nZVI-graphite powder (GP) system to synergistically enhance denitrification under C/N = 3. The results showed that nZVI enhanced nitrate removal efficiency without affecting the removal rate, but excessive dose showed marginal improvement due to the inhibitory effect. In contrast, nZVI coupled GP further enhanced removal performance, increasing nitrate removal efficiency by up to 45.2% at 14 h compared to nZVI. The ferrous ion experiment indicated that released ion did not account for the denitrification enhancement. While nZVI elevated cell biomass, it concurrently induced oxidative stress and redirected metabolic electrons toward polyhydroxybutyrate (PHB) synthesis and cell proliferation rather than denitrification. In contrast, by optimizing electron flux, nZVI coupled GP effectively reduced PHB-associated electron loss while amplifying NADH regeneration (1.4-fold) to fuel denitrification. Furthermore, nZVI coupled GP established redox homeostasis by activating glycine-mediated antioxidant pathways to mitigate reactive oxygen species (ROS) accumulation induced by nZVI. The nZVI-GP system effectively balanced microbial growth and enzymatic needs while optimizing electron distribution, overcoming limitations of nZVI. Gene expression analysis revealed that nZVI coupled GP regulated the genes related to carbon metabolism (e.g. zwf, fumB), amino acid/biotin synthesis (e.g. soxA, bioB), and iron transporters (e.g. hmuU, hemN), enhancing the utilization of carbon source, micro-nutrient uptake, which were key pathways for denitrification. This biocompatible approach improves nitrogen removal in carbon-limited wastewater, offering a promising hybrid treatment solution.