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.