Redox-Related Metabolic Dynamics Imprinted on Short-Chain Carboxylic Acids in Soil Water Extracts: A ¹³C-Exometabolomics Analysis

Soil microbial metabolism is critical to carbon cycling, but direct annotation of metabolic activities is challenging. Here, we present an exometabolomics approach coupled with 13C profiling for in situ probing of metabolic activities following a 13C-glucose pulse in oxic or anoxic soil incubations. In the soil water extracts, we monitored both abundance and isotopic enrichment of short-chain carboxylic acids (SCCAs) involved in different metabolic pathways: peripheral sugar oxidation, glycolysis, fermentation, and the tricarboxylic acid cycle. The water-extractable SCCAs captured redox-dependent metabolic dynamics. First, under both redox conditions, increased concentration (up to 50 μM) along with near-complete 13C-labeled fractions for both gluconate and 2-ketogluconate revealed activation of the peripheral sugar oxidation pathway. Second, greater citrate depletion during the oxic condition than during the anoxic condition (6.2 versus 2.3 μM) was consistent with an expected decrease in carbon usage in the anoxic incubations. Third, accumulation of 13C-labeled succinate and malate only in the anoxic incubations demonstrated metabolic overflow typical of anaerobic metabolism. Fourth, production of 13C-labeled lactate under the anoxic conditions highlighted glucose fermentation but, under the oxic conditions, lactate depletion implied its role as a carbon source. Such 13C-assisted exometabolomics data offer insights to interpret gene-based predictions of metabolic potentials.