Phosphorus limitation constrains microbial carbon–nitrogen cycling in a Eucalyptus forest under elevated CO2: evidence from EucFACE

Phosphorus (P) can restrict the capacity of forests to store additional carbon (C) with increasing carbon dioxide (CO₂) concentration. Although P limitation is widespread, P addition experiments in mature forests are rare, leaving large uncertainties about whether alleviating P limitation under elevated CO₂ (eCO₂) will enhance C storage or instead shift limitation toward nitrogen (N). Here, we used a parallel P-fertilization x eCO₂ manipulation in a mature forest to investigate the acute nutrient cycling response to P fertilization under eCO₂ with a particular focus on N cycling. In April 2023, a mature P-limited Eucalyptus Forest at the Euc-Free Air CO₂ Enrichment (Euc-FACE) experiment in Australia, was fertilized with 1.5 g P m^-2 following 10 years of CO₂ enrichment. We measured soil gross N mineralization and compound-specific depolymerization rates – offering novel insights into microbial metabolic pathways – alongside extracellular enzymatic activities, and nutrient pools in the top 10 cm of soil before P addition, 10 days and two months afterwards. We found that P addition decreased extracellular soil enzymatic activities associated with C-N-P-mining (─ 50%), increased microbial NH₄⁺ retention (immobilization: mineralization ratio; + 23%) and microbial C use efficiency (CUE; + 12%), causing a reduction in plant-available N (─ 30%) independently from eCO₂. Under eCO₂, P addition stimulated protein depolymerization and C-P enzyme activities. Compound specific analyses revealed increased microbial biosynthesis with P addition via the assimilation of key amino acids such as alanine, glycine and glutamate. These findings indicate that P limitation constrains microbial C-N cycling under eCO₂ by diverting microbial C investment toward P acquisition rather than growth. While alleviating P limitation can rapidly stimulate microbial cycling and promotes microbial C retention under eCO₂, this response may only be transient, as enhanced microbial growth drives the system towards N limitation.