Root exudation increases the concentration of readily available carbon (C) compounds in its immediate environment. This creates ‘hotspots’ of microbial activity characterized by accelerated soil organic matter turnover with direct implications for nutrient availability for plants. However, we still lack a deeper understanding of the microbial metabolic processes that occur in the immediate vicinity of the roots during and after a root exudation event. Even though theoretical concepts have been developed, the direct consequences of root exudation on microbial metabolism and nutrient availability have never been measured in their immediate environment in intact soil. Here, we used reverse microdialysis to simulate root exudation by releasing a 13C-labelled mix of low-molecular-weight organic C compounds at discrete, mm-sized locations in undisturbed soil in combination with 13C stable isotope tracing. This approach allowed us to investigate the fine-scale temporal and spatial response of microbial metabolism and soil chemistry to root exudation at the mm-scale, and to trace microbial respiration and uptake of exuded compounds. Our results show that a 9-hour simulated root exudation pulse leads to i) a large local respiration event and ii) alteration of the temporal dynamics of soil metabolites over the following twelve days right at the spot of exudate release. Notably, we observed an approximately threefold increase in ammonium concentrations twelve hours after the pulse and increased nitrate concentrations five days after the pulse. We also observed an increase of various short-chain fatty acids, such as acetate, propionate and formate over the following days, indicating altered microbial metabolic pathways and activity. Phospholipid and neutral lipid fatty acids (PLFAs and NLFAs) of all major microbial groups were significantly enriched in 13C within a radius of 5 mm around the microdialysis probes, but not beyond. The highest relative 13C enrichment was observed in fungal NLFAs, indicating that a significant proportion of the exuded compounds had been incorporated into fungal storage compounds. Our findings indicate that the punctual release of low-molecular weight organic C compounds into intact soil significantly changes microbial metabolism and activity in its immediate surroundings, which lead to enhanced mineralisation of native organic nitrogen (N). Our observations emphasise the versatility of microbial metabolic pathways that underlie the response of soil microbes to rapidly altered C availability. They furthermore demonstrate the effectiveness of this response, as triggered by root exudation pulses, to increase nutrient availability for plants around the root.

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