A Combined Application of Molecular Microbial Ecology and Elemental Analyses Can Advance the Understanding of Decomposition Dynamics

Introducing animal carbon-source to soil initiates biochemical and microbial processes that lead to its decomposition and recycling, which subsequently cause successional shifts in soil microbial community. To investigate the use of soil microbial community to inform criminal investigation, this study was designed to mimic clandestine graves. It compared the decomposition of stillborn piglets (Sus scrofa domesticus), as human analogues, to oak (Quercus robur) leaf litter and soil-only controls outdoors for 720 days. Environmental and edaphic parameters were monitored and showed soil microbial community alignment with temperature seasonality, which highlighted the importance of this abiotic factor. Denaturing gradient gel electrophoresis (DGGE) data were used to calculate Hill numbers and diversity indices of the bacterial 16S rRNA community did not distinguish mammalian- from plant-based decomposition consistently during the first or second year of the study. In contrast, the fungal 18S rRNA community allowed clear differentiation between different treatments (beta diversity) throughout the 720-day experiment and suggested the moment of the decomposing mammalian skin rupture. 16S rRNA-based NGS facilitated the identification of e.g., Pirellulaceae, Acidobacteria ii1-15_order and Candidatus xiphinematobacter as Year 2 bacterial markers of gravesoil at family, order and species taxonomic levels, respectively, and confirmed the similarity of the calculated Hill diversity metrics with those derived from DGGE profiling. Parallel soil elemental composition was measured by portable X-ray Fluorescence where calcium profiles for the piglet-associated soils were distinct from those without carrion. Also, soil calcium content and PMI correlated positively during the first year then negatively during the second. This study is one of the first to apply a multidisciplinary approach based on molecular and physicochemical analytical techniques to assess decomposition. It highlights the recognised potential of using soil microbial community in forensic investigations and provides a proof-of-concept for the application of a combined molecular and elemental approach to further understand the dynamics of decomposition. In addition, it sets the scene for further research in different conditions based on Hill numbers metrics instead of the classic ecological indices for soil necrobiome richness, diversity and evenness.