Microbial nitrogen use efficiency (NUE) reflects the partitioning of organic N taken up between N incorporation into microbial biomass (growth) and N recycled to the environment as inorganic N (mineralization). It is an important parameter to understand N retention and availability, and soil organic matter (SOM) stability. Enhancing SOM content through climate smart practices is one way to mitigate effects of climate change and improve soil health. Whole orchard recycling (WOR), which is the on-site grinding and incorporation of tree biomass into soil prior to replanting an orchard, is one such climate smart practice that has strong potential to be carbon (C) negative and build SOM. Prior studies on greenhouse gas (GHG) fluxes and soil nutrient dynamics indicate there is a shift in the stability of OM in WOR soils over time. The role of microorganisms in mediating GHG fluxes and in C and N transformations in recycled soils remains largely unknown. This proposal aims to use metabolomics and metatranscriptomics in conjunction with quantitative measurements of microbial NUE, gross N mineralization rates, and nitrous oxide fluxes to discern the molecular controls of microbial N uptake in recycled soils. Using stable isotope probing/tracing experiments, we will test the overall hypothesis that recycled soils will have higher microbial NUE than control soils and that N fertilization rates will determine the predominant N uptake pathways. This proposed research would be a continuation of ongoing work at EMSL that aims to show detection of 15N-labeled metabolites via solution state NMR in soil extracts. This work is relevant to BER’s Biological Systems research and Earth and Environmental Systems research missions by providing a mechanistic understanding of microbial processes involved in nutrient cycling and C sequestration in agricultural soil systems.