Conference Agenda

The availability of soil nutrients for plants is dominated by the internal recycling of the nutrient within the ecosystem via mineralization. Understanding and quantifying mineralization in soil is, hence, crucial for understanding ecosystem functioning and production. The actual dynamics of mineralization and other nutrient transformations is given by their gross rates, which cannot be measured directly but are quantified using an isotopic tracer in conjunction with kinetic modelling. This is one of the most powerful tools within biogeochemistry and has been applied for terrestrial nutrient cycling (mostly nitrogen) since the 1950’s.

The use ¹⁵N labelling techniques (pool dilution and tracing) to quantify gross nitrogen turnover in soils is widespread today. However, many researchers applying these techniques are seemingly not aware of critical assumptions and limitations of the ¹⁵N labelling techniques, which unnecessarily compromises our understanding of ecosystem nitrogen dynamics. Also, differences in applied experimental protocols might hinder meaningful meta-studies on controlling factors for gross N transformations, if these methodological differences are not considered in interpretation.

In this presentation, I will give a historic review on the ¹⁵N labelling methodologies for quantification of gross nutrient transformation rates and revisit the underlying assumptions. Based on examples and literature review we will discuss important knowledge gaps that future research should address. Moreover, we will discuss the need of standardization of experimental protocols of ¹⁵N labelling studies for gross N rates determination, to allow better comparison and synthesis of gross nitrogen dynamics in soils. On the long-term, this will ultimately improve our understanding of ecosystem nutrient biogeochemistry and, hence, ecosystems functioning.

Nitrous oxide (N₂O) emissions contribute significantly to greenhouse gas effect and are mainly produced through agricultural practices, particularly following the application of nitrogen-based fertilizers. The isotopic composition of N₂O can provide useful information for assessing N₂O sources. However, due to the co-occurrence of multiple N₂O microbial transformation pathways, it is challenging to use isotopic information to quantify the contribution of distinct processes. Nevertheless, identifying this is responsible for N₂O emissions is crucial to better understand the underlying mechanisms and develop targeted climate change mitigation strategies.

In recent decades, the analysis of abundance of the four most abundant isotopocules of N₂O (¹⁴N¹⁴N¹⁶O, ¹⁴N¹⁵N¹⁶O, ¹⁵N¹⁴N¹⁶O, ¹⁴N¹⁴N¹⁸O) has been a promising tool to evaluate N₂O production pathways (heterotrophic bacterial denitrification, nitrifier-denitrification, fungal denitrification, nitrification) and N₂O reduction to N₂. To obtain a best estimate for N₂O reduction, this approach can be combined with the ¹⁵N gas flux method with N₂-depleted atmosphere (¹⁵NGF⁺), which allows direct quantification of N₂ based on ¹⁵N tracing. Nevertheless, the N₂O isotopocule approach cannot distinguish between heterotrophic bacterial denitrification and nitrifier denitrification, while the ¹⁵NGF⁺ method cannot differentiate between nitrifier-denitrification and nitrification, but between fungal denitrification/heterotrophic bacterial denitrification and nitrifier denitrification/nitrification. Thus, the combination of both approaches provides values for heterotrophic bacterial denitrification and nitrifier-denitrification and thus improves our understanding of the N₂O processes.

We will present the calculation strategies and examples of combined datasets of N₂O isotopocules and ¹⁵NGF⁺ from various laboratory and field studies.

Alpine lakes are increasingly impacted by global environmental changes. Specifically, changing nutrient (nitrogen (N) and phosphorus (P)) inputs are of significant concern for the functioning of these ecosystems^[1]. Long range transport from anthropogenic sources, such as agriculture and fossil fuel combustion, is an important nutrient source to alpine regions via atmospheric deposition^[2]. However, local sources (e.g. glacial meltwaters, tourism) could increasingly subsidize nutrients to alpine lakes ^[3],[4]. This raises the question: which one of these changing sources predominantly drives nitrogen and phosphorus cycling in alpine lakes?

Eight lakes in the French Alps were selected along a gradient of anthropogenic and environmental pressures. Samples from the water column and sediment were taken over 3 seasons (winter, spring, summer) to trace the origin and fate of nutrients in the lakes. More specifically, stable isotope analysis is being conducted on nitrate (δ¹⁵N, δ¹⁸O and ∆¹⁷O), ammonium (δ¹⁵N) and phosphate (δ¹⁸O). Preliminary results show that nitrate isotopic composition in water and sediment samples is similar regardless of the lake. This suggests that either all the lakes have a common driving source of nitrate, or that nitrate is biologically cycled in or before reaching the lakes. Combining these results with ongoing ammonium and phosphate isotopy analysis will give a novel multi-dimensional framework to interpret nutrient cycling in alpine lakes.

[1] Oleksy et al., (2021), Freshwater Science, DOI : 10.1086/713068.

[2] Galloway et al., (2003), BioScience, DOI : 10.1641/0006-3568.

[3] Saros et al., (2010), Environmental Science and Technology, DOI : 10.1020/es100147j.

[4] Baron et al., (2023), Ecosphere, DOI : 10.1002/ecs2.4504.

This study explores nitrogen transformations in groundwater from an agricultural area utilizing organic fertilizer (wastewaters from yeast production) integrating isotopic analysis, microbial gene abundance, and the FRAME (isotope FRactionation And Mixing Evaluation) model to trace and quantify nitrogen cycling pathways (1). Groundwater samples with elevated nitrate concentrations were subjected to controlled laboratory incubations with application of a novel low-level ¹⁵N tracing strategy, to investigate microbial processes. Isotope analyses of nitrate, nitrite and N₂O, coupled with microbial gene quantification via qPCR (2), revealed a shift from archaeal-driven nitrification to bacterial denitrification post-incubation in suboxic conditions, stimulated by glucose addition. FRAME modeling further identified bacterial denitrification (bD) as the dominant pathway of N₂O production, which was supported by increased nosZI, nirK and nirS gene abundance and observed isotope effects.

Simultaneously to the intensive nitrate reduction, it was observed that the majority of nitrite is likely produced through nitrification processes linked to dissolved organic nitrogen (DON) oxidation. Nitrate reduction had minor contribution in the total nitrite pool. The results demonstrate the efficacy of integrating multi-compound isotope studies and microbial analyses to unravel nitrogen cycling mechanisms. This approach provides a robust framework for addressing nitrogen pollution in groundwater systems and improving water quality management strategies.

References:

1.Lewicki et.al.,. FRAME—Monte Carlo model for evaluation of the stable isotope mixing and fractionation. PLoS One. 2022;17(11):e0277204.

2.Deb et.al.,. Microbial nitrogen transformations tracked by natural abundance isotope studies and microbiological methods: A review. Science of the Total Environment. 2024:172073.