Plant wax n-alkanes trapped in soil humin by non-covalent bonds

Soil organic matter is a very complex mixture of compounds derived from the decay of numerous living organisms. Earlier reports suggest that soil organic molecules may occur unaltered in a "bound" form, e.g. by encapsulation in the humic substances matrix or by binding involving weak forces such as H bonds and Van der Waals forces. This issue is relevant to the release of pesticides and other toxic compounds in waters during several years, which suggests a particular mechanim of storage of organic compounds in soil organic matter. However, so far, the mechanim of weak binding is poorly known due to possible analytical bias in the isolation of "weakly-bound" molecules, and to the lack of analytical approaches that can distinguish a "free" compound from its "weakly-bound" counterpart. Here, we analysed the 13C isotope composition of plant-derived soil n-alkanes in a soil sample from an experimental field cropped 23 years with maize to label soil organic carbon. Indeed, cultivating maize, a C4 plant with 13C-enriched carbon, on a soil previously cropped with C3 plants, introduces 13C-enriched carbon in the soil. Thus maize-derived soil carbon can be distinguished from previously-cropped plant soil carbon by 13C analysis. We used a recently developped analytical technique, gas chromatography - isotope ratio monitoring mass spectrometry (GC-IRMS), that allows to measure the 13C isotope ratio of individual substances occurring in complex mixtures. We analysed three pools of C27-C33 n-alkanes in the same soil sample: 1) "free" n alkanes that were extracted with CHCl3-MeOH, 2) "bound" n-alkanes that were extracted from humin, the macromolecular part of soil organic matter, and 3) "bound" n-alkanes that were released by pyrolysis of the pre-extracted humin. Our results show that free n-alkanes are 13C-enriched by +5.8-7.0‰ versus their bound n-alkanes counterparts. This finding have several implications. First, this clear isotope difference of the the same substance occurring either in free or bound form proves that there is no analytical bias such as a lack of exhautive extraction. Second, the higher 13C content of free n-alkanes evidences their higher turnover versus bound n-alkanes. Here a first-order kinetic law yields an age difference of 7 years between free and bound n-alkanes. Third, since n-alkanes are apolar compounds our results demonstrate the existence of a mechanism of weak binding involving either encapsulation or weak forces. This mechanism could explain why chemicals such as pesticides are well preserved in soil then released in waters several years after the end of their use in cropping systems. It is also relevant to the storage of organic-N compounds as possible plant nutrient precursors.