Microbial processing of fresh carbon inputs is recognized as a key step in the formation of mineral-associated organic matter. Low molecular weight (LMW) compounds comprise a notable fraction of these inputs and are rapidly assimilated and metabolized by the microbial community. In this work, we employ ecophysiological studies of microbial isolates to better understand the role of substrate identity as a control on preferences, uptake kinetics, and carbon use efficiencies (CUE) across a gradient of phylogenetic differences (gram negative, gram positive, and fungal). Soil-extracted, solubilized organic matter (SESOM) derived from the Oa horizon of a hemlock-hardwood forest stand and synthetic media based off of this extract were chosen as liquid media for batch growth studies. A combination of exometabolomic techniques (1H NMR, UHPLC-MS) were used to quantify 35 LMW substrates in the original extract (0.4 – 195 μM), comprising 19.5% of total C and 39.9% of total N. Consumption of these substrates by microbial isolates accounted for a substantial amount of total C and N assimilated during growth, representing 43-75% and 58-74%, respectively. Time resolved sampling allowed modeling of sigmoidal uptake curves and the comparison of the midpoint of consumption (Th, hr) and 90% usage windows (ranging from 0.18 – 2.29 hr). Complementary experiments were conducted using synthetic media with all substrates at equimolar concentrations (25 μM) to better constrain the impact of initial concentration. We use stable isotope probing to determine CUE for five different LMW substrates of interest (glucose, acetate, formate, glycine, and valine). Ultimately, we are interested in whether unifying trends can be observed across the physiological gradient and how the metabolic transformations of these inputs may impact the organo-mineral formation process.