Long-Term Nitrogen Addition Reshapes Methane and Nitrous Oxide Fluxes and Microbial Functional Potential in European Beech Forest Soil

European beech (Fagus sylvatica L.) is a both native and extensively cultivated species found in Central and Southeast Europe's upland forests. These beech forests soils are known to emit nitrous oxide (N₂O), sequester methane (CH₄), and release carbon dioxide (CO₂), individually influenced by specific site conditions. The interplay of nitrogen (N) and carbon cycling, along with greenhouse gas (GHG) turnover in these forests, is affected by N deposition, but the long-term effects of N-deposition on GHG exchange involving soil and mature trees are not well understood.

We examined how simulated increased N-deposition affects GHG emissions and soil N-composition in a pre-alpine eutrophic beech forest in Northeastern Italy, subjected to high N-addition. Since 2015, the site has undergone N-manipulation involving four treatments with three replicates: control (N0, ambient N-deposition), above canopy N-addition (N30A, 30 kg/ha*yr N), and soil N-addition at 30 and 60 kg/ha*yr, respectively (N30 and N60). For this study, one plot for each treatment was considered. In September 2023, we measured N₂O, CH₄, and CO₂ fluxes from stems and accompanying soil, and analyzed soil samples for biological and physico-chemical properties.

Beech stems acted as net CH₄ sinks and CO₂ sources, with limited N₂O exchange, unaffected by nine years of artificial N-treatment. Similarly, soil CO₂ emissions remained unchanged, but soil CH₄ uptake increased by 40% in N30 and N60 plots. Conversely, N-treated plots showed significantly lower soil N₂O emissions than controls (nearly 50-fold difference). High flux variability suggests that the observed effects cannot be solely ascribed to N-treatment, likely due to the influence of complex micro-topography.

Soil analyses revealed that N-addition strongly affected soil chemistry, and microbial functional diversity. Control plots maintained higher concentrations of nitrate, nitrite, and total dissolved inorganic nitrogen, indicating enhanced N-consumption or transformation rates under elevated inputs. The N-addition reorganized the microbial community, marked by increased richness and evenness and a shift towards reductive processes, confirmed by the enrichment of genes associated with assimilatory and dissimilatory nitrate reduction and denitrification. Furthermore, carbon cycle responses included increased methanotrophic capacity in N60, evidenced by pmoA gene enrichment, while this effect was absent in canopy-applied treatments.

Overall, while long-term N-addition did not significantly alter GHG stem fluxes, it facilitated greater soil CH₄ uptake through increased microbial methane oxidation capacities and caused substantial restructuring of microbial communities with increased N-reduction potential.

This research was supported by the Ministry of Education, Youth and Sports of CR within the programs LU-INTER-EXCELLENCE II [LUC23162] and CzeCOS [LM2023048], project AdAgriF-Advanced methods of greenhouse gases emission reduction and sequestration in agriculture and forest landscape for climate change mitigation [CZ.02.01.01/00/22_008/0004635], and by the Spanish Government grants PID2024-162617NB-I00 funded by MCIN, AEI/10.13039/ 501100011033 EU Next Generation EU/PRTR