Isotopic overprinting of nitrification on denitrification PNAS PLUS as a ubiquitous and unifying feature of environmental nitrogen cycling Julie Grangera,1 and Scott D. Wankelb,1 aDepartment of Marine Sciences, University of Connecticut, Groton, CT 06340; and bDepartment of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02540 Edited by Donald E. Canfield, Institute of Biology and Nordic Center for Earth Evolution (NordCEE), University of Southern Denmark, Odense M., Denmark, and approved August 26, 2016 (received for review January 25, 2016) 15 Natural abundance nitrogen and oxygen isotopes of nitrate (δ NNO3 invaluable tool to differentiate sources, track their distribution, 18 and δ ONO3) provide an important tool for evaluating sources and and determine the biogeochemical transformations acting on − − transformations of natural and contaminant nitrate (NO3 )inthe NO3 . By convention, isotope ratios are reported using δ notation, − 15 15 14 15 14 18 environment. Nevertheless, conventional interpretations of NO3 where δ N = ([ N/ N]sample/[ N/ N]air − 1) × 1,000 and δ O = 18 16 18 16 isotope distributions appear at odds with patterns emerging from ([ O/ O]sample/[ O/ O]VSMOW − 1) × 1,000, in units of per mille studies of nitrifying and denitrifying bacterial cultures. To resolve (‰). Given two isotopic tracers for a single compound, this this conundrum, we present results from a numerical model of approach can be powerful, as each isotope system provides com- − δ18 NO3 isotope dynamics, demonstrating that deviations in ONO3 plementary information on sources and biogeochemical transfor- 15 vs. δ NNO3 from a trajectory of 1 expected for denitrification are − mations (2). Accurate interpretation of isotope distributions, explained by isotopic over-printing from coincident NO3 production however, strongly hinges on knowledge of the isotope com- by nitrification and/or anammox. The analysis highlights two driving position of source terms and on a rigorous understanding of parameters: (i)theδ18Oofambientwaterand(ii) the relative flux − isotopic discrimination associated with biological transforma- of NO3 production under net denitrifying conditions, whether tions of N pools. The isotopic discrimination associated with SCIENCES catalyzed aerobically or anaerobically. In agreement with existing specific N transformations is quantified by the isotope effect, e, ENVIRONMENTAL > analyses, dual isotopic trajectories 1, characteristic of marine where e (‰) = [(lightk/heavyk) − 1] × 1,000, and k refers to the re- denitrifying systems, arise predominantly under elevated rates of − − > spective specific reaction rate constants of light and heavy iso- NO2 reoxidation relative to NO3 reduction ( 50%) and in associa- topologues (3). Although many of the important source terms and tion with the elevated δ18O of seawater. This result specifically im- − isotope effects of the N cycle are constrained, some remain equiv- plicates aerobic nitrification as the dominant NO producing term in 3 ocal. In particular, recent observations emerging from bacterial and marine denitrifying systems, as stoichiometric constraints indicate − archaeal cultures and from incubations of environmental samples anammox-based NO production cannot account for trajectories − 3 have uncovered isotopic discrimination trends for NO isotopes >1. In contrast, trajectories <1 comprise the majority of model solu- 3 that appear at odds with trends typically ascribed to analogous tions, with those representative of aquifer conditions requiring − biological transformations in soils and aquifers (4–14). This de- lower NO2 reoxidation fluxes (<15%) and the influence of the lower δ18O of freshwater. Accordingly, we suggest that widely ob- velopment has led to conflicting environmental interpretations, 18 15 reflecting a lack of consensus on fundamental isotope systematics of served δ ONO3 vs. δ NNO3 trends in freshwater systems (<1) must − the processes driving the N cycle. Importantly, the discrepancies result from concurrent NO3 production by anammox in anoxic aqui- fers, a process that has been largely overlooked. Significance nitrate | nitrification | denitrification | isotopes | anammox Stable isotopes of nitrate have long provided a tool for track- he advent of the Haber–Bosch process late in the 19th cen- ing environmental sources and biological transformations. Ttury initiated an unprecedented increase in anthropogenic However, divergent interpretations of fundamental nitrate loading of reactive nitrogen (N) to the biosphere, setting into isotope systematics exist among disciplinary divisions. In an motion cascades of environmental impacts, including eutrophica- effort to transcend disciplinary boundaries of terrestrial and tion and hypoxia, ecosystem acidification, and loss of biodiversity marine biogeochemistry, we use a quantitative model for (1). This intensification of environmental N release from agricul- coupled nitrogen and oxygen isotopes of nitrate founded on tural and industrial activities, power generation, municipal and benchmarks established from microbial cultures, to reconcile septic wastewater, and domestic fertilizer has tremendously al- decades of nitrate isotopic measurements in freshwater and tered the global N cycle, effectively doubling annual global N seawater and move toward a unified understanding of cycling turnover (1). In groundwater, the most common nitrogenous processes and isotope systematics. Our findings indicate that − denitrification operates within the pervasive context of nitrite contaminant is nitrate (NO3 ), with recognized and long-term effects on both human and ecological health. Thus, control and reoxidation mechanisms, specifically highlighting the relative − importance of nitrification in marine denitrifying systems and elimination of NO3 contamination are priorities of environ- mental and health agencies worldwide. Despite its significance to anammox in groundwater aquifers. − global health and ecosystem function, identifying sources of NO3 , tracing its dispersal and attenuation, and gauging its ecological Author contributions: J.G. and S.D.W. designed research, performed research, analyzed data, and wrote the paper. impact remain challenging. − The authors declare no conflict of interest. Mitigation of NO3 pollution has necessitated identification of its sources and hydrologic flow paths to monitor the fate and This article is a PNAS Direct Submission. 1To whom correspondence may be addressed. Email: [email protected] or sdwankel@ natural attenuation processes occurring in pollutant plumes. To whoi.edu. this end, the natural abundance stable isotope ratios of nitro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 15 14 18 16 − gen ( N/ N) and oxygen ( O/ O) in NO3 have provided an 1073/pnas.1601383113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1601383113 PNAS | Published online October 4, 2016 | E6391–E6400 Downloaded by guest on September 26, 2021 between isotopic trends in environmental systems and those from freshwater environments (39, 40), although this tenet has not been culture-based observations raise the possibility that biogeochemical examined specifically. The potential for analogous biogeochemical − N dynamics inferred from environmental NO3 isotopic measure- dynamics to affect isotope distributions in freshwater and marine ments reflect more complexity than previously realized. systems clearly merits exploring. − − Conventional interpretation schemes for NO3 isotopes differ To arrive at a shared understanding of environmental NO3 from culture observations with regard to the isotope systematic of isotope systematics, we present the results of a multiprocess nu- − − − denitrification, the stepwise reduction of NO3 to NO2 (nitrite), merical model of dual NO3 isotope dynamics parameterized on nitric oxide, nitrous oxide, and finally N2 by heterotrophic bacteria, the basis of fundamental features revealed from culture studies. which is the dominant loss term of reactive N from the biosphere. From this improved understanding of isotopic fractionation during − − Early studies of NO3 isotope dynamics in groundwater docu- redox cycling of N, we explore implications for NO3 production – 15 18 − − mented parallel enrichment of δ Nandδ OofNO3 in associa- by NO2 oxidizing bacteria and by anammox—occurring concur- − − tion with NO3 attenuation from denitrification, approximating a rently with denitrification, specifically focusing on resulting NO3 N linear trajectory with a slope 0.5–0.8 (15–17). Indeed, this salient and O isotope trajectories. We use this framework to evaluate the − trend has long been considered a unique diagnostic signal of de- potential extent of processes other than unidirectional NO3 con- nitrification (2). However, following the advent of the denitrifier sumption by denitrification, which may harbor the key for resolving − method (18, 19), measurements in cultures of both freshwater and the discrepancy between decades of groundwater NO3 observa- marine denitrifying bacteria revealed dual isotope enrichments as- tions and our physiological understanding of the isotope systematics − sociated with assimilatory and dissimilatory NO3 consumption of microbial N cycling. The scenarios explored herein call attention systematically following linear trajectories of ∼1 (9, 10, 12, 19, 20), to the potential influence of N cycling dynamics that have been contrasting with the lower values widely observed in freshwater