Short communication
Microbial characteristics of soils from graves: an investigation at the
interface of soil microbiology and forensic science
D.W. Hopkins
a,∗, P.E.J. Wiltshire
b, B.D. Turner
caDepartment of Environmental Science, University of Stirling, Stirling FK9 4LA, Scotland, UK bInstitute of Archaeology, University College London, 31-34 Gordon Square, London WC1H 0PY, UK cDivision of Life Sciences, King’s College, University of London, 150 Stamford Street, London SE1 8WA, UK
Received 16 January 2000; received in revised form 16 March 2000; accepted 16 March 2000
Abstract
Very little is known about the microbiology of graves. We have taken the opportunity to investigate this subject by taking advantage of the unusual opportunity afforded by the experimental burial of pigs in a forensic experiment. Selected microbial characteristics of soils from the 0–15 and 15–30 cm depths of the graves of three pigs and of control soils have been determined 430 days after burial. The grave soils contained more total C, microbial biomass C and total N, and showed increased rates of respiration and N mineralisation compared to the control soils. The grave soils also had larger amino acid and NH4+ concentrations, which was consistent with the increases in both net N mineralisation and pH values. Nitrification was not detected in any of the soils and the limited NO3−supply restricted the rate of denitrification, but the large alkali-soluble S2− concentration of soils from the graves indicated reducing conditions in the graves. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Biomass; Bodies; Carcass disposal; Decay; Denitrification; Sulphur reduction
1. Introduction
The expertise of archaeologists with skills in exca-vating human remains is sometimes harnessed to help in the investigation of fatalities under suspicious cir-cumstances (Hunter et al., 1996). Whilst the opportu-nities to study the decomposition of human remains are obviously limited, some information is available from war crime investigations (Mant, 1987). In order to collect evidence for a murder investigation, it is
∗Corresponding author. Tel.:+44-1786467851;
fax:+44-1786467843.
E-mail address:[email protected] (D.W. Hopkins)
sometimes necessary experimentally to bury and then monitor the decay of pigs’ carcasses as analogues of a human victim. The evidence from such investigations often takes the form of a qualitative description of the state of the body’s decay with details of any inverte-brates that colonised the decaying flesh (Erzinclioglu, 1983).
Details of the microbial processes in the decom-posing body are rarely determined. Janaway (1996) describes the succession from predominantly aerobic to anaerobic members of the enteric community act-ing alongside autolysis duract-ing cadaveric decay. The study presented here is a microbiological investigation of the soils from the graves of three pigs buried in a
forensic experiment allied to a genuine murder inves-tigation. In this particular case, the burial experiment was necessary because the body of the human vic-tim was considered to be particularly well preserved for the suspected post-mortem interval and other at-tempts to establish the period of burial had been in-conclusive. However, the results we present here were collected after the conclusion of the forensic inves-tigation and have no bearing on the murder enquiry. We have taken advantage of the unusual opportunity that the experiment afforded us to undertake a de-tailed investigation of the microbiology of soil from graves.
2. Materials and methods
2.1. Burial experiment and soils
The carcasses of three 4 to 5-month old pigs were buried within 3 h of death beneath 10 cm of soil in a
Carpinus betulus(hornbeam) dominated woodland in Hertfordshire, England in late December 1996. The burial sites were between 5 and 20 m from the grave of a human murder victim that had been discovered in April 1996. The pigs’ graves were formed by re-moving the litter layer, excavating the soil, and then replacing most of it on top of the pigs’ bodies to leave a grave level with the surrounding ground sur-face which was then covered with leaf litter. The soils were stagnogleys of the Essenden association formed in drift and tertiary clays. Scavengers (probably foxes or badgers) discovered all the graves at various times. Disturbance was greatest for the grave of Pig 2, which was partially opened twice, and least for the grave of Pig 1, the surface of which was disturbed once, but without any part of the pig’s corpse being ex-posed. On each occasion when the graves were dis-turbed they were repaired as far as possible by refilling with soil. Soil samples were collected during February 1998, 430 days after burial. Control soils were col-lected from sites approximately 1 m away from each of the graves. By the time the samples were taken, the pigs’ bodies had lost their integrity and the graves con-tained mixtures of decaying remains and soil. Sam-ples from depths of 0–15 and 15–30 cm were taken by careful excavation at several locations close to the original position of the trunk of each pig avoiding as
far as possible sites of disturbance. The soils were sieved (7 mm) in the field-moist state and large stones and fragments of roots were removed, as were two vertebrae from the 15–30 cm sample from grave of Pig 1.
The soil moisture content was determined by dry-ing at 105◦C. The soil C and N contents were
de-termined using a Carlo-Erba CHN analyser. Soil pH was determined on a 1:2.5 w/v suspension in distilled water. NH4+, NO3− and a-amino-N (amino acids) were determined on soil extracts prepared in 0.5 M K2SO4using, respectively, the indophenol-blue,
sali-cylic acid-NaOH methods described by Anderson and Ingram (1993) and the ninhydrin method described by Rowell (1994). For estimation of amino acids, the ninhydrin assay result was corrected for the presence of NH4+. The soil microbial biomass was determined
using the glucose induced respiration (CO2
produc-tion) approach of Anderson and Domsch (1978), with slight modifications described by Hopkins and Fergu-son (1994) in which CO2was determined by gas
chro-matography (thermal conductivity detector). Basal respiration was estimated from soil incubated in absence of added glucose.
N mineralisation rates were estimated from the amount of NH4+ that accumulated during 7 days
incubation at 37◦
C under anaerobic conditions (ap-proximately 2 g fresh weight soil) in 10 cm3 water, as described by Keeney (1982). Short-term nitrifier activity was determined in duplicate from the NO2−
that accumulated when soil (approximately 2 g fresh weight samples) was shaken in 20 cm3 of a solution
of 0.5 mmol (NH4)2SO4and 10 mmol NaClO3dm−3
for 24 h at 20◦C as described in Hopkins et al. (1988).
The denitrification rate in the soil was determined by incubating soil (approximately 10 g fresh wt.) for 24 h at 20◦C in the respirometric devices used for CO
2
measurements, the head-spaces of which were filled with air, and C2H2 at a partial pressure of 10 kPa,
and determining the N2O that accumulated by gas
chromatography (thermal conductivity detector). The potential rates of denitrification were determined by shaking soil (approximately 3 g fresh wt.) for 24 h at 20◦
C with 10 cm3 of distilled water, 10 cm3 of 50 mmol NaNO3 dm−3 solution, or 10 cm3 50 mmol
3. Results
3.1. Variability between the graves
In several respects samples from the three experi-mental replicates behaved differently from each other and the data from each of them have not, therefore, been combined. Disturbance of the graves by scav-engers was a possible reasons for this (Table 1).
3.2. Chemical and physical properties
For all three controls, the soil C and N contents were greater at the surface than at depth, reflecting the distribution of leaf litter (Table 1). The total C con-tent from upper sample from control 2 was consiscon-tent with it apparently being mostly leaf litter. By contrast, there was significantly more C and N at depth for the less-disturbed Grave 1; no significant difference in C but more N at depth for Grave 2; and more C near the surface than at depth for the Grave 3, although the difference in N content with depth was not significant (Table 1). Despite the differences in C and N distribu-tion with depth in the graves, there was more C and N at depth in all of the graves than in any of the control soils (Table 1). The most obvious factor contributing to the differences in C and N distribution is the pres-ence of the pigs remains. The soils NH4+and amino
acid concentrations were all greater at the surface than at depth for the controls and reversed, i.e.greater con-centrations at depth than at the surface, for the graves. The pH of soil from the controls was between pH 3.5 and 3.8, whilst it was between 3.8 and 5.6 for the grave soils. In the graves of Pigs 1 and 2, the pH was greater at 15–30 cm than at 0–15 cm, whilst for the grave of Pig 3, there was no difference with depth. The highest pH values coincided with large NH4+concentrations.
3.3. Microbial biomass and activity
In every case (graves and controls), the biomass C was greater for the 0–15 cm depth than for the 15–30 cm depth, although the difference with depth for the disturbed graves of Pigs 2 and 3 was not sig-nificant (Table 1). For the controls, the biomass values were consistent with the total C contents (Table 1), the biomass C being generally greater for samples with
larger C contents. For all three graves, the biomass C contents of the 15–30 cm samples were greater than the 15–30 cm samples of the controls.
The respiration rates were also greater at 0–15 cm than at 15–30 cm for all three controls and greater at depth than the surface for all three graves (Table 1). For samples from the graves of Pigs 1 and 3 (i.e. the less disturbed graves), the respiration rates at 0–15 cm were greater than Controls 1 and 3. In the case of the 0–15 cm sample from Control 2, which had a very large C content due to disturbance, the respiration rate was greater than that of the 0–15 cm sample from the grave.
N mineralisation showed a similar pattern to the res-piration rates, with greater values for the graves com-pared with the controls and increasing with depth in the graves (Table 1). The N mineralisation for Con-trol 2 was apparently influenced by its large organic N content. The N mineralisation rates were consistent with the NH4+ and amino acid distributions (Table
1), and suggest that the greater pH of soil from the graves was due to NH4+ release during
ammonifica-tion of the protein and other organic N sources from the decaying pig.
With the exception of the sample from the 0–15 cm depth of Control 2, the soils from the controls were grey, yellow and brown in colour (Table 1). The sam-ple from the surface of Control 2 was dark in colour, which is consistent with it being primarily leaf lit-ter. By contrast, the soils from the graves were pre-dominantly grey and olive (greenish) in colour. This predominance of grey and olive colours is consistent with the concentrations of S2− in the grave samples, the concentrations of which for the upper samples were greater than those at either depth in the controls (Table 1). These data indicate that substantial S re-duction had occurred and a predominance of reducing conditions in the graves. The rates of denitrification were, however, very small for all the soils and only detectable with acceptable accuracy (using a gas chro-matograph fitted with a thermal conductivity detector) for the samples from the grave of Pig 1. Results from the experiment to test the effects of additional NO3−,
and NO3−plus glucose on samples from control and
Table 2
Effect of nitrate addition and nitrate plus glucose addition on the denitrification rate of grave and control soils from Pig 1a
Depth (cm) Denitrification rate (nmol N2O g−1 soil h−1)
Unamended Nitrate addition Nitrate and glucose addition
Grave 0–15 0.18 (0.010) 1.90 (0.68) 6.1 (0.23)
15–30 0.35 (0.030) 4.7 (0.72) 46.7 (3.10)
Control 0–15 0.13 (0.0050) 0.081 (0.024) 0.14 (0.052)
15–30 0.12 (0.0050) 0.079 (0.029) 0.090 (0.0023)
aEach value is the mean of three replicates and the standard deviations are shown in brackets.
glucose addition were small and not significant for the control soils, but led to significant increases in deni-trification rate for the grave samples. Even under the influence of large NH4+concentrations, the soils from
the graves were acidic and the short-term nitrifier ac-tivities were all below 0.1 (mg N g−1soil h−1(data not shown)), which was, in effect, the limit of detection using the procedure described. This is consistent both with the effect of NO3−addition on denitrification and
the fact that only traces of NO3−were detected in the
samples (i.e. in the range 0–0.5mg NO3−-N g−1soil;
data not shown).
4. Discussion
This study is an opportunity seized at the end of an-other experiment and this has imposed limitations on it. However, in the absence of other soil microbiolog-ical studies of graves, we have produced potentially useful preliminary information of interest to archae-ologists, forensic scientists and those concerned with disposing of the carcasses of farm animals. The na-ture of the original forensic experiment restricted the sampling so that soil could only be collected after the forensic objectives had been fulfilled. Important parts of the decomposition process will have occurred in the 430 days before the samples were taken. Nevertheless, the elevated NH4+ concentrations, biomass and
res-piratory activity all indicate that decomposition was still taking place and the elevated S2− concentrations reflect the occurrence of S reduction in the period before the samples were taken.
The observations that addition to soil of a large amount of relatively readily decomposable organic resource with a heavy microbial inoculum in the form of the enteric community led to increased microbial biomass and activity is not in itself surprising. This
study has provided data that accounts, in part at least, for the good state of preservation of the human body when discovered. The burial conditions, in poorly draining soil, are likely to have contributed to the soil conditions becoming anoxic following the addition of the pigs bodies. In addition, the intense acidity of the soil will have led to a restriction in the supply of NO3−, from nitrification, as an alternative electron
acceptor to O2 for the decomposer organisms, and
probably to a more general restriction in soil micro-bial activity. At the stage when the graves and control soils were sampled for this study, substantial amelio-ration of the soil pH had occurred as a result of NH4+
released from the pigs. At early stages in the experi-ment, field measurements of soil pH were as high as 7.2. Despite this, and the ample supply of NH4+, the
wet, acidic soil was still unfavourable for nitrification. The study has provided a snap-shot of decomposition processes in a particular set of graves and many ques-tions are left unanswered. For future work in this area it will be important to establish the extent to which soil conditions affect the decomposition of human bodies and to improve our understanding of the processes earlier in decay. For example, had the pigs (or human victim) been buried in near-neutral soil or in fertilised agricultural fields, would the supply of NO3− have
been sufficient to avoid a restriction on microbial respiration? Alternatively, had the pigs (or the human victim) been buried in better drained soils, how much, if any, faster would they have decomposed?
Acknowledgements
insti-tutions are duly acknowledged, as is the Hertfordshire Constabulary.
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