Nitrogen availability alters the contribution of ex-vivo and in-vivo pathways to carbon formation in particulate- and mineral-associated organic matter

• The contribution of ex-vivo pathway to POM-C increased with root biomass and rhizodeposited C. • Nitrogen availability increased microbial incorporation of the rhizodeposits. • Nitrogen availability increased contribution of the in-vivo pathway to MAOM-C. • The ex-vivo and in-vivo pathways co-contributed to the formation of POM-C. Soil carbon (C) sequestration depends on the formation of particulate- and mineral-associated organic matter (POM and MAOM). These soil organic matter (SOM) pools are largely derived from rhizodeposition, but how the quality and quantity of rhizodeposits affect the direct contribution of root-derived C ( ex-vivo pathway) and microbially processed root-derived C ( in-vivo pathway) to the formation of each pool remains unclear. To test this, we induced variation in quality and quantity of plant C inputs by imposing defoliation frequencies (3–4 vs . 6–8 clipping events year −1 ), and varying N fertiliser (0 vs . 40 kg N ha −1 yr −1 ) application rate for five years in a grassland. Two 2-hour 13 CO 2 pulse labelling events were applied in mid- and late summer to estimate the short-term incorporation of root-derived C in SOM pools. Water soluble metabolites in soil and near edge X-ray absorption fine structure (NEXAFS) spectroscopy of POM and MAOM fractions were used to characterise the organic C. Results showed that N fertilisation increased microbial utilisation of the rhizodeposited C and the contribution of in-vivo pathway to MAOM-C, as supported by increased concentration of trehalose (microbe-derived metabolite) associated with MAOM-C. Moreover, N fertilisation increased POM-C accumulation via ex-vivo pathway, as shown by increased abundance of carboxylic C groups, likely reflecting increased fine root biomass and rhizodeposited C. At the same time, N fertilisation increased the abundance of O -alkyl C functional groups in POM-C fraction that are mostly microbially driven likely suggesting that decaying microbes or mycorrhizal hyphae also contribute to POM-C highlighting the co-contribution of the in-vivo pathway with N addition. Moreover, both ex-vivo and in-vivo pathways contributed into MAOM-C at low rhizodeposition rate (e.g. in highly defoliated treatment particularly when no N was applied) as shown by increased abundance of the aromatic C with side chain and N-substituted aromatic C and increased concentration of metabolites such as malic acid and mannose, which can originate from both plants and microbes. Our results shed light on how variation in root biomass, rhizodeposition, and N availability affects the contribution of ex-vivo and in-vivo pathways in the formation of root-derived C in POM and MAOM fractions.