Phosphorus supply and spatial distribution determine the microbial response to carbon and nitrogen addition

• Response to C and N addition was largely consistent for topsoil and subsoil samples. • C and N addition enhanced enzymatic mineralization of organic P in low P soils. • Microscale microbial activity is enhanced in patches rich in organic matter and P. Phosphorus (P) supply can limit microbial activity especially under conditions of high carbon (C) and nitrogen (N) supply, but this observation is mostly based on analyses of topsoil bulk samples, while processes in the subsoil or at the microscale remain largely unexplored. Here, we collected topsoil (0–30 cm) and subsoil (>30 cm) samples from two temperate forest and two arable sites (Cambisols and Luvisols with pH 4.2 to 7.0 and C content of 2 to 113 g kg −1 ), to provide a set of model soil samples with varying P content and varying contributions of organic and mineral P forms. We conducted an incubation experiment at 20 °C for 16 weeks with addition of either glucose (C treatment), ammonium nitrate (N treatment), both glucose and ammonium nitrate (CN treatment), or without additions (control). In a separate incubation of topsoil samples with CN addition and 18 O labeled water, we analyzed microsite enrichment with 18 O as an indicator of microbial activity using nanoscale secondary ion mass spectrometry (NanoSIMS). Soil respiration rates were significantly increased after C and CN addition but not after N addition, and organic P mineralization by acid phosphatase was also enhanced with CN addition. However, the response in respiration rates after CN addition in topsoil samples decreased with increasing soil C:P ratio, while acid phosphatase activity increased, indicating increasing P limitation and mineralization of organic P in samples with low P supply (wide C:P ratio). In the subsoil samples, the response in microbial and enzymatic activity was weaker, but showed a similar trend as in topsoils. NanoSIMS analyses indicated 18 O enrichment especially in P-rich microsites of soils with low P content and in co-location with patches dominated by organic matter. We conclude that also at the microsite level, under conditions of high C and N supply, microbial activity is controlled by the inherent supply of P in both arable and forest soils studied here.