Tracing the Mineralisation Rates of C, N and S from Cysteine and Methionine in a Grassland Soil: A <sup>14</sup>C and <sup>35</sup>S Dual-Labelling Study

Cysteine (Cys) and Methionine (Met) represent the two main sulphur (S)-containing amino14 acids found in soil solution. Although general measures of S cycling (e.g., sulfatase activity) provide valuable information concerning the cycling of labile organic S in soil, detailed information regarding the microbial transformation pathways of Cys and Met at a molecular level remain poorly characterised. Therefore, in this study a 14C and 35S dual-isotopic labelling approach was used to trace the fate of C and S derived from Cys and Met in an agricultural grassland soil over a 7-day incubation period. Microbial biomass C, N, and S were analysed by the CHCl3 fumigation-extraction method, and CO2 evolution along with inorganic nutrient release (NH4 + , NO3 - and SO4 2-) were also measured. We then imposed an excess of C as glucose, or excess NPS (as NH4NO3, KH2PO4 and K2SO4) to investigate whether the Cys and Met mineralization process was affected by manipulating C, N and S availability in the soil solution. Our results showed that after 168 h, 2.7 – 19.5% of the 14C derived from Cys and Met had been immobilised in the microbial biomass, 67.2 – 89.2% had been respired as 14CO2 while the recovery of 35S label in the soil microbial biomass ranged from 11.9 – 41.8%. Overall, our results indicated that microbial communities have a high capacity to utilize Cys and Met but that they enter multiple metabolic pathways once inside the cell. While some of the amino acids may be directly used in protein synthesis, other metabolic pathways lead to NH4 + and SO4 2- being released back into the soil with the NH4 + then rapidly converted to NO3 - by the nitrifier community. The resulting C-skeletons were dissimilated and used to produce energy, leading to the release of 14CO2. The significant differences in C, N and S mineralization processing demonstrate a decoupling of the S and C cycles at the molecular level. Further, glucose-C addition shifted the allocation of C inside the cell to more catabolic processes and greater mineralization, while in comparison inorganic N, P and S availability had much less effect on resource partitioning.