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Coupled nitrogen and oxygen isotope fractionation of nitrate imparted during its assimilation and dissimilatory reduction by unicellular plankton Granger, Julie

Abstract

I report the first measurements of coupled nitrogen (N) and oxygen (O) isotopic variations of nitrate (NO₃⁻) during its assimilation and dissimilatory reduction by laboratory cultures of marine and freshwater plankton. I derive the N and O kinetic isotope effects for nitrate assimilation by strains of marine and freshwater phytoplankton, as well as N and O isotope effects for denitrification by marine and freshwater strains of denitrifying bacteria. Large inter- and intra-species variations in the N and O isotope effects were observed among phytoplankton and among denitrifiers. However, the O isotope effect associated with either nitrate consumption or denitrification always co-varied with the N isotope effect, such that the ¹⁸O/¹⁶O and ¹⁵N/¹⁴N of nitrate changed concomitantly with a consistent ratio of ~1:1, regardless of species or culture conditions. A single strain of denitrifiers, Rhodobacter sphaeroides, showed an O-to-N co-variation of 0.6. My results indicate that the dominant driver of the N and O isotope effects is nitrate reductase. The O-to-N ratio of I owes to the isotopic signature of the various nitrate reductases involved in nitrate reduction, namely the eukaryotic assimilatory nitrate reductase (eukNR), the prokaryotic assimilatory nitrate reductase (NAS), as well as the prokaryotic respiratory nitrate reductase (NAR). The variability in the observed magnitude of the N and O isotope effects is attributed to changes in the ratio of cellular nitrate uptake and efflux. The unusual O-to-N ratio of 0.6 observed for R. sphaeroides is imprinted on nitrate by the periplasmic auxiliary nitrate reductase NAP. I also report a novel method to remove nitrite from samples for isotopic analysis of nitrate with the ’denitrifier method,’ which measures the isotopic composition of nitrate and nitrite concomitantly. Nitrite is removed with ascorbate while purging with an inert gas. The method is non-toxic (hence compatible with the denitrifier method) and does not alter the concentration or the isotopic composition of nitrate in the samples. The findings reported here provide insight into the physiological mechanisms underlying nitrate isotopic fractionation during its assimilation and dissimilatory reduction. The trends observed in culture underline the biological systematics from which to interpret in situ measurements of coupled N and O isotopes of nitrate. Moreover, this work emphasizes the need to investigate the environmental factors that potentially contribute to variations in the magnitude of the N and O isotope effects in situ.

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