Awakening a latent phosphoenolpyruvate- oxaloacetate-glyceraldehyde carbon-fixation pathway for cost-effective nitrogen removal by adjusting carbon source and pH in the anammox-centered process

Sustainable development based on carbon fixation is a promising orientation for CO2 emission reduction. Here, in an anammox-centered coupling system that involves partial denitrification and hydrolytic acidification (A-PDHA), a latent carbon-fixation pathway called phosphoenolpyruvate-oxaloacetate-glyceraldehyde (POG) cycle, was firstly awakened through high inorganic carbon injection. Correspondingly, a nitrogen removal efficiency of > 95 % and significant reduction of organic carbon demand were both achieved. To understand the nature of low-carbon and efficient nitrogen removal performance, the carbon fixation mechanism and its driving energy metabolism were elucidated using metaproteomics and metabolomics. Results revealed that the POG cycle was energy-saving, and kinetically and thermodynamically feasible. This autocatalytic route involves the reduction of HCO3− using the most efficient natural phosphoenolpyruvate carboxylase, followed by the phosphorylation of one molecule of glucose from starch, and then the glycolysis pathway. These guarantee the abundant production of carbon fixation products i.e., glyceraldehyde-3-phosphate and acetyl-CoA. Additionally, the neutral pH facilitates these products flowing into the tricarboxylic acid cycle, thereby generating sufficient adenosine triphosphate and reducing power, which further drives downstream multi-pathway nitrogen metabolism. These findings provide valuable insights for future research on artificial carbon emission reduction in anammox-centered coupling systems during wastewater treatment.