Efficient denitrification and N2O mitigation in low-C/N wastewater treatment by promoting TCA cycle anaplerosis via glyoxylate shunt regulation

Conventional biodenitrification for water with a low carbon-to-nitrogen ratio (C/N) demands exogenous carbon, exacerbating carbon consumption and emissions. Here we propose a metabolic reprogramming strategy leveraging Mo(VI)–Fe(III)–Cu(II) synergy to redirect carbon flux through the glyoxylate shunt (GS), enhancing tricarboxylic acid cycle anaplerosis for efficient denitrification and reduced greenhouse gases during low-C/N wastewater treatment. At a C/N of 3, Mo(VI)–Fe(III)–Cu(II) promoted carbon metabolism by the tricarboxylic acid cycle in Paracoccus denitrificans, elevating reducing power (electron carriers) production and electron transporter activity. This increased total nitrogen removal by 196.2% compared with the blank control and by approximately 32.0–146.6% compared with single- or dual-metal-supplemented controls, while reducing nitrous oxide emissions by 51.3% and approximately 26.2–85.6%, respectively. This effect originated from the inhibition of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase by Mo(VI)–Fe(III)–Cu(II), causing isocitrate accumulation that activates isocitrate lyase of the glyoxylate shunt and prioritizes GS-driven anaplerosis. Finally, activated sludge validation increased 31.7% total nitrogen removal efficiency, highlighting the approach’s practical potential. This carbon-metabolism reprogramming strategy reduces organic carbon demand in denitrification, enhancing energy efficiency and advancing carbon-neutral wastewater treatment. This study proposes a strategy for enhancing denitrification in low-C/N wastewater by redirecting carbon flux through glyoxylate shunt regulation.