Isotopic fractionation of N and O in N2O from denitrification: insights from the comparative analysis of Pseudomonas strains with distinct nitrite reductase enzymes

Nitrous oxide (N2O), a potent greenhouse gas, primarily stems from oxidative (e.g. nitrification) and reductive (e.g., denitrification) microbial processes in aquatic and terrestrial environments. To better understand spatial and temporal N2O production, the isotopic composition of N2O, specifically 15N/14N and 18O/16O ratios, and the intramolecular distribution of 15N (i.e., site preference, SP), are typically used[1]. Distinguishing multiple concurrent processes with three isotope parameters (δ15N, δ18O, SP) remains, however, a challenge, especially in light of uncertainties regarding the isotope effects for individual processes. Here, we study the isotope effects of N2O production imparted by denitrification, focusing specifically on the intermediate step of nitrite (NO2-) reduction to nitric oxide (NO), which is catalyzed by various nitrite reductases. We study three bacterial denitrifiers: Pseudomonas chlororaphis subsp. aureofaciens, Pseudomonas chlororaphis, and Pseudomonas stutzeri. These bacteria utilize similar nitric oxide reductases (NorB) enzymes, but different nitrite reductase variants (NirS vs. NirK). We anticipate similarities in SP values, mostly controlled by NorB, but differences in δ15N-bulk and δ18O values for generated N2O, given the distinct nitrite reductase enzymes[2]. P. stutzeri strain JM300, expressing both NirS and NirK genes[3], offers a unique opportunity for studying each enzyme's distinct functions and isotopic signatures. Selected strains are incubated in batch experiments of a 0.5 L bioreactor using nitrate as a substrate. The bioreactor's headspace is continuously purged with N2. We monitor bacterial growth, NO2- concentrations, dissolved O2, pH, and temperature throughout the experiment. Simultaneously, we daily collect one sample for nitrite and nitrate N and O isotope analysis. After removing CO2 and water, N2O concentrations are monitored with Fourier-transform infrared spectroscopy. The isotopic composition of N2O is measured online using quantum-cascade-laser spectroscopy, providing real-time analysis with high precision (< 0.1 ‰). This enables real-time tracking of changes in the N and O isotope systematics (i.e., fractionation), in response to changing reaction kinetics. The preliminary data that we present will lay the basis for future investigations into the constraints on systematic heavy-isotope clumping (i.e., relative abundance of doubly substituted N2O isotopologues 15N15N16O, 14N15N18O, 15N14N18O) associated with microbial N2O production. Specifically, we will verify direct and indirect enzymatic controls (i.e., type of Nir; N-O bond equilibration with water) on the clumped-isotope abundance of 14N15N18O.   [1] Toyoda, S., et al. (2017). Isotopocule analysis of biologically produced nitrous oxide in various environments. Mass Spectrometry Reviews, 36(2), 135-160. [2] Martin, T. S., et al. (2016). Nitrogen and oxygen isotopic fractionation during microbial nitrite reduction. Limnology and Oceanography, 61(3), 1134-1143. [3] Wittorf, L., et al. (2018). Expression of nirK and nirS genes in two strains of Pseudomonas stutzeri harbouring both types of NO-forming nitrite reductases. Research in microbiology, 169(6), 343-347.