Reverse Microdialysis and Isotope Labelling Reveal Microbial Strategies for Carbon Storage in the Rhizosphere

Root exudates are key drivers of microbial activity and carbon cycling in the rhizosphere, but their transient and localised nature makes microbial responses hard to measure. We used reverse microdialysis to deliver glucose as a root exudate analogue and trace microbial synthesis and carbon allocation.  Deuterated water (D₂O) quantified baseline rates of synthesis in unamended soil, while ¹³C-glucose traced microbial synthesis fuelled by C from localised glucose input.  Over 72 hours, we quantified incorporation of ²H and ¹³C into metabolites, membrane lipids, and storage compounds. Glucose perfusion significantly increased microbial respiration and synthesis rates, particularly for polyhydroxybutyrate (PHB) and triacylglycerols (TG), indicating strong stimulation of intracellular carbon storage. The rapid incorporation of ²H and ¹³C into diacylglycerols (DGs), coupled with slow turnover, suggests DGs may function in intracellular carbon storage or as membrane lipids rather than solely as transient metabolic intermediates.  Glucose perfusion also increased membrane lipid synthesis, with differences in ¹³C incorporation among membrane lipids indicating differential growth among microbial groups.  In contrast to larger increases in synthesis of intracellular C storage and membrane lipids, synthesis and turnover of compatible solutes such as trehalose and mannitol were largely unaffected by glucose perfusion, implying their roles are independent of carbon supply and tied to metabolic regulation in well-watered soil. Our results highlight the utility of reverse microdialysis and dual isotope labelling for disentangling effects of root exudates on microbial metabolism. This approach provides new insights into how localized carbon inputs shape microbial function and community dynamics, and emphasises intracellular carbon storage as a key microbial response for coping with transient resource availability in the rhizosphere.