PHA stimulated denitrification through regulation of preferential cofactor provision and intracellular carbon metabolism at different dissolved oxygen levels by Pseudomonas stutzeri

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Denitrification, a typical biological process mediated by complex environmental factors, i.e., carbon sources and dissolved oxygen (DO), has attracted great attention due to its contribution to the control of eutrophication and the biochemical cycling of nitrogen. However, the effects of carbon source on electron distribution and enzyme expression for enhanced denitrification under competition of electron acceptors (DO and nitrate) remain unclear. Here, we profile the carbon metabolic pathway of polyhydroxybutyrate (PHB) and glucose (Glu) at high and low DO levels (50% and 10% saturated DO, respectively). It was found that PHB enhanced the growth of Pseudomonas stutzeri (model denitrifying bacterium) and improved the specific nitrogen removal rate (SNRR) at all DO levels. The functional proteins had a better affinity for the cofactor nicotinamide-adenine dinucleotide (NADH) than for nicotinamide adenine dinucleotide phosphate (NADPH); thus, more electrons were involved in nitrogen reduction and intracellular PHB production in the PHB groups than in the Glu groups. Furthermore, the expression difference of enzymes in glucose and PHB metabolism was demonstrated by metaproteomic and target protein analysis, implying that PHB-driven intracellular carbon accumulation could optimize the intracellular electron allocation and correspondingly promote nitrogen metabolism. Our work integrated the mechanisms of intracellular carbon metabolism with preferences for electron transfer pathways in denitrification, providing a new perspective on how the selective parameters regulated microbial functions involved in denitrification.