Further optimisation of the denitrifier method for the rapid ¹⁵N and ¹⁸O analysis of nitrate in natural water samples

Abstract

Funding information Deutsche Bundesstiftung Umwelt (DBU) Rationale: This study aims to develop a simplified denitrifier method for the δ 15 N and δ 18 O analysis of nitrate (NO 3 −) in natural water samples combining the method of Zhu et al (Sci Total Environ. 2018; 633: 1370-1378) and the original denitrifier method of Sigman et al (Anal Chem. 2001; 73: 4145-4153). Unlike in the aforementioned methods, the aerobic cultivation was performed without the addition or removal of nitrate in the liquid medium. We remove the nitrate contained in the nutrient medium as N 2 O in the gas phase by an additional purging step after incubation overnight before the water sample is injected. This eliminates the need for another preparation step, thus saving working time. Methods: The δ 15 N and δ 18 O values of dissolved NO 3 − were determined using a Delta V Plus isotope ratio mass spectrometer coupled to a GasBench II sample preparation device that included a denitrification kit. Results: After optimising the influencing factors (i.e., purging gas, purging time, and type of crimp seals), the method yielded high accuracy and precision (standard deviations were generally ≤0.7‰ for δ 18 O values and ≤0.3‰ for δ 15 N values), confirming the suitability of this procedure. Finally, the potential applicability of the method was demonstrated by measuring the isotopic composition of NO 3 − in natural water samples. Conclusions: The denitrifier method for converting NO 3 − into N 2 O for isotope analysis was optimised. This allowed the sample preparation time to be further reduced. The complete working time for sample preparation, including all steps, takes 10 min per vial if 60 vials are prepared in one run. The water samples are ready for isotope analysis on the fourth day after preparation has started. Isotope measurements can be performed up to 14 days after preparation. 1 | INTRODUCTION Understanding the geochemical processes that occur in the subsoil is necessary for risk assessments of groundwater and the protection of water resources. The identification of NO 3 − sources and the quantification of NO 3 − degradation in groundwater are particularly important. 1 As NO 3 − from different sources, such as fertiliser, wastewater, or animal waste, tends to exhibit characteristic isotopic compositions, its origin can be inferred by measuring the ratios of the heavy to light isotopes of nitrogen and oxygen. 2-4 In addition, biological processes can change these isotope ratios. Thus, isotope analysis facilitates the study of these processes 2 and is frequently used in the environmental sciences. 3 To determine the isotopic composition of NO 3 − in aqueous samples, it must first be

Comparison of Denitrification Induced by Various Organic Substances—Reaction Rates, Microbiology, and Temperature Effect

“…Isotope characterization of N and O in dissolved NO 3 − (Delta V Plus isotope ratio mass spectrometer, TU Dresden) was conducted for the acetate column tests only, whereby samples were stored until analysis at −20°C. The analysis was performed with microbial transformation from NO 3 − to N 2 O according to the denitrification method of Zhu et al (2018), which was further developed by Stock et al (2020). Notation is expressed in δ‰ (δ = ([R sample − R standard ]/R standard ), where R is the ratio between the heavy and the light isotopes).…”

“…(2018), which was further developed by Stock et al. (2020). Notation is expressed in δ‰ (δ = ([ R sample − R standard ]/ R standard ), where R is the ratio between the heavy and the light isotopes).…”

Challenges in measuring nitrogen isotope signatures in inorganic nitrogen forms: An interlaboratory comparison of three common measurement approaches

“…However, if NO 2 − is detectable in the samples, it needs to be removed by sulfamic acid or ascorbic acid pretreatment 51,62,63 . However, high salt concentrations should not be a problem for the N 2 O‐based methods, as supported by published data sets on reference materials prepared in seawater and freshwater (e.g., references 28 and 51 ) but it can be a problem for DN and CM‐N 2 64,65 …”

“…51,62,63 However, high salt concentrations should not be a problem for the N 2 O-based methods, as supported by published data sets on reference materials prepared in seawater and freshwater (e.g., references 28 and 51) but it can be a problem for DN and CM-N 2 . 64,65 Other method comparisons for δ 15 N of NO 3 À in environmental samples showed much better (though also not optimal) agreement between different methods tested, for example, groundwater and surface water samples differed only by 0.4-0.5‰ between MD and CM-N 2 in a study carried out by Sebilo et al 66 Thus, it shows that high accuracy can be achieved with MD if sufficient care is taken to optimize the MD procedure (Figure 3). Similarly, there was sufficient agreement (Δδ 15 N of 1.5‰) between δ 15 N of NO 3 À of surface waters measured by a CM-N 2 (AgNO 3 ) method and DN, though much larger differences were found for specific samples.…”

Vertical Nitrate Migration And Denitrification Zones In The Regional Recharge Area (Lwowek Region, Poland)

“…Dissolved nitrate was transformed completely into gaseous N2O by a culture of the denitrifying bacterium Pseudomonas aureofaciens. The used, simplified denitrification method was published by (Stock et al 25 ). After microbial sample preparation, simultaneous δ15N and δ18O analyses of N2O sample gases were performed by a GasBench II device with a denitrification kit and a Delta V Plus isotope mass spectrometer (both Thermo Fisher Scientific).…”