Evaluating concentration-dependent effects of deuterated water to optimize its use as marker of metabolic activity in soil microbiomes

Deuterated water (²H₂O) has been used to investigate changes in the metabolic activity of microorganisms. In contrast to, for example, ¹³C-labeled compounds, ²H₂O acts as a general marker of biosynthetic activity, does not alter the available substrate pool and is more cost-effective than ¹⁸O-labeled water. These properties make ²H₂O an attractive alternative for stable isotope labeling experiments, ranging from small-scale microcosm incubations with only a few grams of soil to larger-scale and more integrative experimental setups. However, high concentrations of deuterium (²H), introduced via ²H₂O, can be toxic to cells, as kinetic isotope effects slow biochemical reaction rates and may therefore inhibit metabolic processes. Consequently, ²H₂O-concentration-dependent effects on metabolic activity in the soil microbiome must be investigated to obtain reliable results. In this study, we conducted a microcosm experiment to analyze the effects of different ²H₂O concentrations (0, 10, 20, 30, 40, 50, 60 at% of ²H) on nitrogen assimilation in the soil microbial community, using ¹⁵N-labeled ammonium sulfate as a tracer. Nanoscale Secondary Ion Mass Spectrometry will be used to derive the metabolic activity of single cells based on the amount of ¹⁵N tracer assimilated at the different ²H₂O concentrations.  Furthermore, metagenomics and metaproteomics will reveal ²H₂O-induced shifts in bacterial community composition and functional pathways. Together, these data will provide the range of ²H₂O concentrations that ensure the non-inhibited metabolic activity in the soil microbiome, supporting its use as a marker in soil microbiome research.