BackgroundMain transformation pathways of low molecular weight organic substances (LMWOS) are understood, but only limited knowledge exists on their transformations in different soils and on their interactions with biochar.AimObjectives were to study short‐term pathways of 13C‐labelled LMWOS with different functional groups in the presence and absence of biochar in arable loess soils.MethodsSoils from three sites were incubated with or without artificially aged biochar and 13C‐labelled acetate, alanine or glucose at different rates (10 or 50 µmol C g−1 soil) at 60% water holding capacity and 15°C for 5 days, and total and substrate‐derived CO2‐C and microbial biomass C (MBC) were determined and analyses of variance were calculated.ResultsCumulative CO2‐C (ΣCO2‐C) emission was significantly (p ≤ 0.05) affected by substrate rate and type and their interaction. Biochar significantly stimulated total, but not substrate‐derived ΣCO2‐C (ΣCO2‐CSD) emission. Box‐Cox transformed MBC was significantly affected by site, substrate rate and type, whereas biochar had no significant effect. Substrate‐derived MBC (MBCSD) and carbon use efficiency (CUE) were significantly affected by site, substrate rate and type and their interaction.ConclusionsMBCSD and CUE results at low addition rate confirmed the greater importance of glucose for the build‐up of MBC compared to acetate and alanine, whereas the latter were mineralized to a greater extent. Biochar, once it is aged, which is the typical biochar form in soil, did not significantly affect build‐up of MBCSD and CUE and only slightly increased total, but not ΣCO2‐CSD emission.