Separation of drilosphere, rhizosphere and bulk soil OM using linear discriminant analysis

The proportions of unsaturated and dicarboxylic FA differed between drilosphere, rhizosphere and bulk soil OM. Therefore, a combination of unsaturated FA and dicarboxylic acids was assumed to improve the separation of OM between these soil compartments. A linear discriminant analysis that was applied to the FA fingerprint of drilosphere, rhizosphere and bulk soil OM revealed a clear separation of the soil compartments.

The separation of bulk soil, rhizosphere and drilosphere OM by the linear combination of relative contents of unsaturated FA and dicarboxylic acids was not affected by soil depth (Figure 2.2-6). Despite differences in the relative contents of unsaturated FA increased with depth, they were lowest in bulk soil, intermediate in rhizosphere and highest in drilosphere in every soil depth (Figure 2.2-4). The same applied to the relative contents of dicarboxylic acids, but the soil compartments differed in reverse order. The lack of a depth effect enabled the utilization of the relative contents of unsaturated and dicarboxylic FA of every soil depth for the discriminant analysis. This indicated depth independency of these FA proxies for OM separation.

The back-tracing of the separation to specific processes could determine, if the combination of unsaturated and dicarboxylic FA yields appropriate molecular markers to differentiate between bulk soil, rhizosphere and drilosphere OM. The effective separation between rhizosphere and drilosphere OM by function 1 is assumed to result from the higher dicarboxylic acid content in the rhizosphere and the lower content of unsaturated FA in the rhizosphere compared to the drilosphere. In arable soil, dicarboxylic acids can trace root-derived organic matter (Mendez-Millan et al., 2010b). Dicarboxylic acids are constituents of suberin that mainly occurs in roots in herbaceous plants (Kolattukudy, 1981; Mendez-Millan et al., 2010b). Degradation of suberin releases dicarboxylic acids into soil. Therefore, higher dicarboxylic acid contents in the rhizosphere compared to the drilosphere could have been caused by the higher amount of root litter in the rhizosphere (Figure 2.2-4). In contrast to dicarboxylic acids, the relative amount of unsaturated FA was lower in the rhizosphere compared to the drilosphere (Figure 2.2-4). It was shown that the relative contribution of

unsaturated compounds to plant biomass was much higher than to soil organic matter (Mendez-Millan et al., 2010b; Nierop et al., 2003). Due to the double bond, unsaturated compounds are preferentially degraded in soil compared to saturated compounds (Mendez-Millan et al., 2010b; Nierop et al., 2003). Organic material that passed the gut of an earthworm was shown to be already partially stabilized (Marhan et al., 2007). Consequently, the higher relative amount of unsaturated FA in drilosphere was assumed to result from a higher input of fresh OM and an increased protection against microbial degradation compared to the rhizosphere. The improved separation of bulk soil OM from rhizosphere and drilosphere OM due to linear discriminant function 2 is assumed to reflect the longer degradation process that reduced the relative unsaturated FA content and increased the content of more recalcitrant dicarboxylic acids (Mendez-Millan et al., 2010b).

2.2.5 Conclusions

Currently, the identification of potential places of carbon allocation in soils is one of the major tasks in soil organic matter research. As biopores were identified as one potential pathway to sequester carbon in subsoil horizons, the source apportionment and fate of biopore carbon might be a key to improve our understanding on carbon cycling and carbon sequestration in subsoils. Thus, the aim of the current study was to determine the carbon concentrations in walls of biopores of known origin, i.e. earthworm- or root-derived to trace carbon incorporation via pore systems and another aim was to identify the potential of FA as biomarkers for source apportionment of organic matter in biopores. Carbon significantly accumulated in the biopore walls compared with bulk soil in different soil horizons with higher values for earthworm- than for root-derived pores. This highlights the potential of pore systems to contribute to carbon incorporation especially in carbon-depleted subsoils.

However, further research is required to determine the long-term fate of this incorporated carbon for tracing the sequestration potential. To study pore systems in soils in the long term it is also a prerequisite to know the biogenic origin of the carbon in pore walls, which might be traced by biomarker approaches. Here, the combination of unsaturated and dicarboxylic acids enabled the separation of bulk soil, rhizosphere and drilosphere OM. We could show for the first time that the relative amounts of unsaturated FA and dicarboxylic acids differed between bulk soil, rhizosphere and drilosphere OM but did not change with depth. Therefore, separation of bulk soil, rhizosphere and drilosphere OM in other ecosystems can be possible.

A universal application of the linear combination of relative contents of unsaturated FA and dicarboxylic acids to categorize OM of unknown origin into bulk soil, rhizosphere and drilosphere organic matter requests the evaluation of the discriminant model with samples from other sites and ecosystems as well as a validation using e.g. biopore OM of different origin (including mixed sources) produced under controlled conditions.

2.2.6 Acknowledgements

We highly acknowledge the support of this study by the German Research Foundation (DFG) within the DFG Research group 1320 “Crop Sequences and the Nutrient Acquisition from the Subsoil”. We are grateful to an anonymous reviewer and the editor for constructive comments on the manuscript.