Nitrogen Isotope Fractionation During Archaeal Ammonia Oxidation: Coupled Estimates From Measurements of Residual Ammonium and Accumulated Nitrite.

Maria Mooshammer,Barbara Bayer,Wolfgang Wanek,Lara Jochum,Michaela Stieglmeier,Simon K.-M R Rittmann,Ricardo J E Alves,Michael Melcher,Margarete Watzka,Gerhard J Herndl,Christa Schleper

doi:10.3389/fmicb.2020.01710

Abstract

The naturally occurring nitrogen (N) isotopes, 15N and 14N, exhibit different reaction rates during many microbial N transformation processes, which results in N isotope fractionation. Such isotope effects are critical parameters for interpreting natural stable isotope abundances as proxies for biological process rates in the environment across scales. The kinetic isotope effect of ammonia oxidation (AO) to nitrite (NO2–), performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is generally ascribed to the enzyme ammonia monooxygenase (AMO), which catalyzes the first step in this process. However, the kinetic isotope effect of AMO, or εAMO, has been typically determined based on isotope kinetics during product formation (cumulative product, NO2–) alone, which may have overestimated εAMO due to possible accumulation of chemical intermediates and alternative sinks of ammonia/ammonium (NH3/NH4+). Here, we analyzed 15N isotope fractionation during archaeal ammonia oxidation based on both isotopic changes in residual substrate (RS, NH4+) and cumulative product (CP, NO2–) pools in pure cultures of the soil strain Nitrososphaera viennensis EN76 and in highly enriched cultures of the marine strain Nitrosopumilus adriaticus NF5, under non-limiting substrate conditions. We obtained εAMO values of 31.9–33.1‰ for both strains based on RS (δ15NH4+) and showed that estimates based on CP (δ15NO2–) give larger isotope fractionation factors by 6–8‰. Complementary analyses showed that, at the end of the growth period, microbial biomass was 15N-enriched (10.1‰), whereas nitrous oxide (N2O) was highly 15N depleted (−38.1‰) relative to the initial substrate. Although we did not determine the isotope effect of NH4+ assimilation (biomass formation) and N2O production by AOA, our results nevertheless show that the discrepancy between εAMO estimates based on RS and CP might have derived from the incorporation of 15N-enriched residual NH4+ after AMO reaction into microbial biomass and that N2O production did not affect isotope fractionation estimates significantly.

Highlights

Knowledge of natural 15N abundances and of nitrogen (N) isotope fractionation effects associated with key microbial N transformation processes has contributed greatly to our understanding of the marine N cycle (Casciotti and Buchwald, 2012; Buchwald and Casciotti, 2013) and of terrestrial gaseous N emissions (Houlton and Bai, 2009), namely atmospheric N2O sources and sinks (Yoshida and Toyoda, 2000), and biological N fixation (Vitousek et al, 2013)
Both N. adriaticus and N. viennensis exhibited 15N isotope fractionation factors based on substrate between 31.9 and 33.1, and based on product between 37.7 and 49.1 (Figures 2A–F)
We found no significant difference between the isotope fractionation factors of the different ammonia-oxidizing archaea (AOA) cultures studied here based on δ15N evolution of the substrate or the product

Summary

Introduction

Knowledge of natural 15N abundances and of nitrogen (N) isotope fractionation effects associated with key microbial N transformation processes has contributed greatly to our understanding of the marine N cycle (Casciotti and Buchwald, 2012; Buchwald and Casciotti, 2013) and of terrestrial gaseous N emissions (Houlton and Bai, 2009), namely atmospheric N2O sources and sinks (Yoshida and Toyoda, 2000), and biological N fixation (Vitousek et al, 2013). ΕCP estimates reflect the combined fractionation effects of the isotope equilibrium between NH4+ and NH3 [NH3, the proposed substrate for ammonia oxidation, is depleted in 15N relative to NH4+ (Hermes et al, 1985)], the AMO-catalyzed reaction, and accumulation of several intermediates derived from subsequent enzymatic processes (Casciotti et al, 2003). ΕCP estimates may be affected by the accumulation of essential intermediates, such as hydroxylamine (NH2OH) and by the production of gaseous N by-products (nitric oxide, NO; and nitrous oxide, N2O), which may represent further 15N fractionation steps This could result in a difference of kinetic isotope effect estimates derived from residual substrate (RS) and CP (Casciotti et al, 2003). Could these “leakage” processes alter CP-based estimates of εAMO, but their different contributions to ammonia utilization and to εCP may underlie the large differences observed in εAMO between ammonia-oxidizing organisms (Mariotti et al, 1981; Yoshida, 1988; Casciotti et al, 2003; Santoro and Casciotti, 2011)

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