The strong greenhouse gas nitrous oxide (N2O) can be emitted from wastewater treatment systems as a byproduct of ammonium oxidation and as the last intermediate in the stepwise reduction of nitrate to N2 by denitrifying organisms. A potential strategy to reduce N2O emissions would be to enhance the activity of N2O reductase (NOS) in the denitrifying microbial community. A survey of existing literature on denitrification in wastewater treatment systems showed that the N2O reducing capacity (VmaxN2O→N2) exceeded the capacity to produce N2O (VmaxNO3→N2O) by a factor of 2–10. This suggests that denitrification can be an effective sink for N2O, potentially scavenging a fraction of the N2O produced by ammonium oxidation or abiotic reactions. We conducted a series of incubation experiments with freshly sampled activated sludge from a wastewater treatment system in Oslo and found that the ratio α = VmaxN2O→N2/VmaxNO3→N2O fluctuated between 2 and 5 in samples taken at intervals over a period of 5 weeks. Adding a cocktail of carbon substrates resulted in increasing rates, but had no significant effect on α. Based on these results – complemented with qPCR and metaproteomic data – we discuss whether the overcapacity to reduce N2O can be ascribed to gene/protein abundance ratios (nosZ/nir), or whether in-cell competition between the reductases for electrons could be of greater importance.@en

Homeostatic control of nitric oxide (NO) at nanomolar concentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase (Nir and Nor). We questioned whether this is sufficient: using the reported substrate affinities for cytochrome cd₁ nitrite reductase (cNor), our model of batch cultures of Paracoccus denitrificans predicted NO concentrations orders of magnitude higher than measured. We rejected a hypothesis that the homeostatic control is due to a negative feedback by NO on the activity of NirS because the inclusion of such feedback resulted in too slow anaerobic growth and N₂ production. We proceeded by determining the kinetic parameters for cNor in vivo by a carefully designed experiment, allowing the estimation of NO concentration at the cell surface while anoxic cultures depleted low headspace doses of NO. With the new parameters for cNor kinetics in vivo {v = v_max/[1 + K₂/(NO) + K₁ × K₂/(NO)²]; v_max = 3.56 fmol NO cell⁻¹ h⁻¹, K₁ < 1 nM, and K₂ = 34 nM}, the model predicted NO concentrations close to that measured. Thus, enzyme kinetics alone can explain the observed NO homeostasis. Determinations of enzyme kinetic parameters in vivo are not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.

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