Environmental predictors impact microbial-based postmortem interval (PMI) estimation models within human decomposition soils.

Microbial succession has been suggested to supplement established postmortem interval (PMI) estimation methods for human remains. Due to limitations of entomological and morphological PMI methods, microbes are an intriguing target for forensic applications as they are present at all stages of decomposition. Previous machine learning models from soil necrobiome data have produced PMI error rates from two and a half to six days; however, these models are built solely on amplicon sequencing of biomarkers (e.g., 16S, 18S rRNA genes) and do not consider environmental factors that influence the presence and abundance of microbial decomposers. This study builds upon current research by evaluating the inclusion of environmental data on microbial-based PMI estimates from decomposition soil samples. Random forest regression models were built to predict PMI using relative taxon abundances obtained from different biological markers (bacterial 16S, fungal ITS, 16S-ITS combined) and taxonomic levels (phylum, class, order, OTU), both with and without environmental predictors (ambient temperature, soil pH, soil conductivity, and enzyme activities) from 19 deceased human individuals that decomposed on the soil surface (Tennessee, USA). Model performance was evaluated by calculating the mean absolute error (MAE). MAE ranged from 804 to 997 accumulated degree hours (ADH) across all models. 16S models outperformed ITS models (p = 0.006), while combining 16S and ITS did not improve upon 16S models alone (p = 0.47). Inclusion of environmental data in PMI prediction models had varied effects on MAE depending on the biological marker and taxonomic level conserved. Specifically, inclusion of the measured environmental features reduced MAE for all ITS models, but improved 16S models at higher taxonomic levels (phylum and class). Overall, we demonstrated some level of predictability in soil microbial succession during human decomposition, however error rates were high when considering a moderate population of donors.

Transient hypoxia drives soil microbial community dynamics and biogeochemistry during human decomposition.

Human decomposition in terrestrial ecosystems is a dynamic process creating localized hot spots of soil microbial activity. Longer-term (beyond a few months) impacts on decomposer microbial communities are poorly characterized and do not typically connect microbial communities to biogeochemistry, limiting our understanding of decomposer communities and their functions. We performed separate year-long human decomposition trials, one starting in spring, another in winter, integrating bacterial and fungal community structure and abundances with soil physicochemistry and biogeochemistry to identify key drivers of microbial community change. In both trials, soil acidification, elevated microbial respiration, and reduced soil oxygen concentrations occurred. Changes in soil oxygen concentrations were the primary driver of microbial succession and nitrogen transformation patterns, while fungal community diversity and abundance was related to soil pH. Relative abundance of facultative anaerobic taxa (Firmicutes and Saccharomycetes) increased during the period of reduced soil oxygen. The magnitude and timing of the decomposition responses were amplified during the spring trial relative to the winter, even when corrected for thermal inputs (accumulated degree days). Further, soil chemical parameters, microbial community structure, and fungal gene abundances remained altered at the end of 1 year, suggesting longer-term impacts on soil ecosystems beyond the initial pulse of decomposition products.

Microbial ecology of vertebrate decomposition in terrestrial ecosystems.

Vertebrate decomposition results in an ephemeral disturbance of the surrounding environment. Microbial decomposers are recognized as key players in the breakdown of complex organic compounds, controlling carbon and nutrient fate in the ecosystem and potentially serving as indicators of time since death for forensic applications. As a result, there has been increasing attention on documenting the microbial communities associated with vertebrate decomposition, or the 'necrobiome'. These necrobiome studies differ in the vertebrate species, microhabitats (e.g. skin vs. soil), and geographic locations studied, but many are narrowly focused on the forensic application of microbial data, missing the larger opportunity to understand the ecology of these communities. To further our understanding of microbial dynamics during vertebrate decomposition and identify knowledge gaps, there is a need to assess the current works from an ecological systems perspective. In this review, we examine recent work pertaining to microbial community dynamics and succession during vertebrate (human and other mammals) decomposition in terrestrial ecosystems, through the lens of a microbial succession ecological framework. From this perspective, we describe three major microbial microhabitats (internal, external, and soil) in terms of their unique successional trajectories and identify three major knowledge gaps that remain to be addressed.

Body Mass Index (BMI) Impacts Soil Chemical and Microbial Response to Human Decomposition.

Microorganisms are key decomposers of vertebrate mortalities, breaking down body tissues and impacting decomposition progress. During human decomposition, both extrinsic environmental factors and intrinsic cadaver-related factors have the potential to impact microbial decomposers either directly or indirectly via altered physical or chemical conditions. While extrinsic factors (e.g., temperature, humidity) explain some variation in microbial response during human decomposition in terrestrial settings, recent work has noted that even under the same environmental conditions, individuals can have different decomposition patterns, highlighting the potential for intrinsic factors to impact microbial decomposers. The goal of this study was to investigate the effects of several intrinsic factors (age, sex, diseases at time of death, and body mass index [BMI]) on chemical and microbial changes in decomposition-impacted soils. In a field study conducted at the University of Tennessee Anthropology Research Facility, soils were collected from the decomposition-impacted area surrounding 19 deceased human individuals through the end of active decomposition. Soil physicochemical parameters were measured, and microbial (bacterial and fungal) communities were assessed via amplicon sequencing. BMI was shown to explain some variation in soil pH and microbial response to human decomposition. Hierarchical linear mixed (HLM) effects models revealed that BMI category significantly explained variation in pH response within decomposition-impacted soils over time (HLM F = 9.647; P < 0.001). Additionally, the relative abundance of soil Saccharomycetes in decomposition soils under underweight donors displayed little to no changes (mean maximum change in relative abundance, +6.6%), while all other BMI categories displayed an increased relative abundance of these organisms over time (normal, +50.6%; overweight, +64.4%; and obese, +64.6%) (HLM F = 3.441; P = 0.11). Together, these results reveal intrinsic factors influencing decomposition patterns, especially within the soil environment, and suggest BMI is an important factor for controlling decomposition processes. IMPORTANCE This work begins to address questions about interindividual variation in vertebrate decomposition attributed to intrinsic factors, that is, properties of the carcass or cadaver itself. Most research on factors affecting decomposition has focused on the extrinsic environment, such as temperature or humidity. While these extrinsic factors do explain some variation in decomposition patterns, interindividual variability is still observed. Understanding how intrinsic factors influence microbial decomposers will help reveal the ecological impacts of decomposition. This work also has forensic applications, as soil chemical and biological changes have been suggested as indicators of postmortem interval. We reveal factors that explain variation in the decomposition environment that should be considered in these estimates. This is particularly important as we consider the implications of variations in human populations due to diet, age, BMI, disease, toxicological loading, etc. on forensic investigations dealing with decomposing remains.

Spatial impacts of a multi-individual grave on microbial and microfaunal communities and soil biogeochemistry.

Decomposing vertebrates, including humans, result in pronounced changes in surrounding soil biogeochemistry, particularly nitrogen (N) and carbon (C) availability, and alter soil micro- and macrofauna. However, the impacts of subsurface human decomposition, where oxygen becomes limited and microbial biomass is generally lower, are far less understood. The goals of this study were to evaluate the impact of human decomposition in a multi-individual, shallow (~70 cm depth) grave on soil biogeochemistry and soil microbial and nematode communities. Three individuals were interred and allowed to decay for four years. Soils were collected from two depths (0‒5 and 30‒35 cm) along linear transects radiating from the grave as well as from within and below (85‒90 cm depth) the grave during excavation to assess how decomposition affects soil properties. Along radiating surface transects, several extracellular enzymes rates and nematode richness increased with increasing distance from the grave, and likely reflect physical site disruption due to grave excavation and infill. There was no evidence of carcass-sourced C and N lateral migration from the grave, at least at 30‒35 cm depth. Within the grave, soils exhibited significant N-enrichment (e.g., ammonium, dissolved organic N), elevated electrical conductivity, and elevated respiration rates with depth. Soil biogeochemistry within the grave, particularly in the middle (30‒35 cm) and base (70‒75 cm depth), was significantly altered by human decomposition. Mean microbial gene abundances changed with depth in the grave, demonstrating increased microbial presence in response to ongoing decomposition. Human-associated Bacteroides were only detected at the base of the grave where anoxic conditions prevailed. Nematode community abundance and richness were reduced at 70‒75 cm and not detectable below 85‒90 cm. Further, we identified certain Plectus spp. as potential indicators of enrichment due to decomposition. Here we demonstrate that human decomposition influences soil biogeochemistry, microbes, and microfauna up to four years after burial.