Determining the impact of cadaver decomposition on soil microbiology and potential applications in forensic science Heloise Breton BSc. MSc. 1 Dr. Shari.

Presentation on theme: "Determining the impact of cadaver decomposition on soil microbiology and potential applications in forensic science Heloise Breton BSc. MSc. 1 Dr. Shari."— Presentation transcript:

1 Determining the impact of cadaver decomposition on soil microbiology and potential applications in forensic science Heloise Breton BSc. MSc. 1 Dr. Shari Forbes1,2 Dr. David O. Carter3 1 University of Ontario Institute of Technology, Oshawa, Canada 2 University of Technology Sydney, Sydney, Australia 3 Chaminade University of Honolulu, Honolulu, Hawaii

2 Cadaver decomposition, Soil microbiology & Forensics
Soil is a complex environment with large microbial communities Potential timeline could be based on soil microbial community dynamics Large microbial loads leach into the environment during cadaver decomposition Changes to the microbial community could enable the detection of clandestine grave Impact on soil moisture, pH, texture, etc.

3 Cadaver decomposition, Soil microbiology & Forensics
WHAT WE KNOW SO FAR... Decomposition associated soils show surges in microbial biomass. Important proliferation of a microbial flora comprising mostly of enteric bacteria. Hopkins et al. (2000) Bacteria from a carcass or cadaver are able to survive in the soil environment. Members of the initial soil microbial community “disappear” with the onset of decomposition and return later on. Parkinson (2009) Is the succession of microbial communities associated with corpse decomposition predictable and potentially useful for estimating the postmortem interval? Do characteristic decomposer communities of bacteria and fungi measurably change the endogenous soil community, enabling detection of clandestine gravesites?

4 Methodology Spring 2011 trial – June to September
Summer 2011 trial – July to October Decomposition facility at UOIT Oshawa, ON Canada 3 decomposition sites 3 control sites Sampling adapted to decomposition rates: Days 0, 2, 4, 6, 8, 11, 14, 17, 20, 27, 34, 41, 48, 62, 97 A total of four studies are being carried out as a part of this research project. The two first of these studies are the ones presented here. The studies took place last year and started on June 2nd and July 21st. This allowed us to compare different temperature trends and their potential impact of the decomposition timeline. The studies use 6 sites for sampling, 3 of which are sites of decomposition where a human analogue, in our case pigs are deposited. The other 3 sites are control sites which are representative of the soil being used for the decomposition sites. Each of these sites are sampled in triplicated giving us 9 decomposition samples and 9 control samples for each day. Sampling was adapted according to the rate of decomposition which meant that samples were taken every other day throughout the fresh, bloat and active stages. Once decomposition appeared to slow down and enter the advanced decay stage sampling occurred every 3 days. Shortly after we moved to sampling on a weekly basis, then a bi-weekly and finally monthly.

5 Methodology Soil moisture pH Microbial activity
Fluorescein diacetate assay (FDA) Enzymatic activity Colorimetric measure To measure microbial activity, we opted to use the fluorescein diacetate assay. It is a quick means of measuring general enzymatic activity in a soil sample. This method has proved useful in detecting fluctuations in microbial activity as a result of various nutriment treatments. Soil microbial activity was measured immediately after collection. To appropriately interpret the changes in soil dynamics being observed, soil moisture and pH were also measured.

6 Methodology Fatty Acid Methyl Ester profiling (FAME)
Extraction of FAMEs from soil samples Adapted from the MIDI® FAME method Gas chromatography - mass spectrometry (GC-MS) Marker fatty acids Bacterial Acid Methyl Ester (BAME) standard Community approach

7 DECOMPOSITION TRENDS Fresh Bloat Active Advanced Dry/Remains
Start day and associated accumulated degree days (ADD) of each stage of decomposition Fresh Bloat Active Advanced Dry/Remains Spring 2011 Day 0 Day 2 29.6 Day Day Day    Summer 2011  55.0 Day 4 107.2 Day 11 325.3 Day 48 706.9

8 DECOMPOSITION TRENDS IMPACT ON SOIL
Impact of decomposition on soil begins with the active decay stage. Discolouration Change in consistency (insect activity) Change in moisture content Smell

9 Microbial Activity Results
Spring 2011 Trial Microbial activity typically higher in decomposition soils Decomposition soil Microbial activity decreases as pH increases Control soil Microbial activity decreases as soil moisture increases

10 Microbial Activity Results
Summer 2011 Trial Significant differences between decomposition and control soils were not seen. Soil microbial communities may be less susceptible to change as a result of decomposition later into the summer season.

11 Microbial Activity Results
No clear trend could be observed from these studies Factors such as pH, soil moisture, temperatures, etc. likely have the most influence on levels of microbial activity Ongoing studies show a tendency towards decomposition inhibiting microbial activity

12 FAME PROFILING RESULTS
Spring 2011 Trial Extracted FAMEs from decomposition soils were more diversified during the active decay stage Oleic acid (C18:1) significantly increased in decomposition soil during active decay Oleic acid (C18:1) -Facultative anaerobic bacteria fatty acid marker

13 FAME PROFILING RESULTS
Spring 2011 Trial Levels of palmitoleic acid (C16:1) and myristic acid (C14:1) aid in distinguishing control soils and decomposition soils. Palmitoleic acid (C16:1) Myristic acid (C14:1) – Anaerobic bacteria fatty acid marker

14 FAME PROFILING RESULTS
Summer 2011 Trial No significant difference between decomposition and control soils prior to day 8 (active decay stage) FAME profiles of decomposition soils in dry/remains resembled those of control soils

15 FAME PROFILING RESULTS
Summer 2011 Trial Extracted FAMEs were more diversified in decomposition soils during active decay . C16:0 (Palmitic acid) showed a significant increase in decomposition soils sampled during active decay C16:0 (Palmitic acid) - Anaerobic bacteria marker fatty acid

16 CONCLUSIONS Naturally occurring changes in soil microbial communities must be better understood The impact of decomposition on soil microbiology is not typically seen until a few days into active decomposition Having a more diversified or more populous microbial community does not translate to increased microbial activity

17 CONCLUSIONS Marker fatty acids showing potential as decomposition identifiers are mainly tied with anaerobic microbial groups Soil profiles showed an increase in facultative anaerobic bacteria which are expected with the decomposition of a body

18 Next steps DNA profiling Extraction - PowerSoil® DNA extraction kits
Amplification – 16S region V4 Sequencing – Illumina® Sequencing by synthesis Analysis – QIIME (Quantitative Insights Into Microbial Ecology)

19 ACKNOWLEDGMENTS Ms. Lori Van Belle Dr. Andrea Kirkwood
Dr. Janice Strap

20 REFERENCES CAVIGELLI, M.A., ROBERTSON, G.P. & M.J. KLUG (1995) Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure. Plant and Soil, 170, p PARKINSON, R. (2009) Bacterial Communities Associated with Human Decomposition, Victoria University of Wellington, Unpublished PhD Thesis PAYNE, J.A. (1965) A Summer Carrion Study of the Baby Pig Sus Scrofa Linnaeus. Ecology 46(5), QUEZADA, M. BUITRON, G. MORENO-ANDRADE, E. MORENO, G. & L.M. LOPEZ-MARIN (2007) The use of fatty acid methyl esters as biomarkers to determine aerobic, facultatively aerobic and anaerobic communities in wastewater treatment systems, Federation of European Microbiological Societies Letters, 266, p.75-82 SANCHEZ-MONDERO, M.A., MONDINI, C., CAYUELA, M.A., ROIG, A. CONTIN, M., & M. DE NOBILII (2008) Fluorescein diacetate hydrolysis, respiration and microbial biomass in freshly amended soils, Biology and fertility of soils, 44(6), p SHERLOCK MICROBIAL IDENTIFICATION SYSTEM (1996) Operating Manual, Version 6, Newark.