Bacterial Communities Associated with Human Decomposition Rachel Parkinson A thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Doctor of Philosophy in Science Victoria University of Wellington 2009 Abstract Human decomposition is a little-understood process with even less currently known about the microbiology involved. The aim of this research was to investigate the bacterial community associated with exposed decomposing mammalian carcasses on soil and to determine whether changes in this community could potentially be used to determine time since death in forensic investigations. A variety of soil chemistry and molecular biology methods, including molecular profiling tools T-RFLP and DGGE were used to explore how and when bacterial communities change during the course of a decomposition event. General bacterial populations and more specific bacterial groups were examined. Decomposition was shown to cause significant and sequential changes in the bacterial communities within the soil, and changes in the bacterial community often correlated with visual changes in the stage of decomposition. Organisms derived from the cadavers and carcasses were able to be detected, using molecular methods, in the underlying soil throughout the decomposition period studied. There was little correlation found between decomposition stage and the presence and diversity within the specific bacterial groups investigated. Organisms contributing to the changes seen in the bacterial communities using molecular profiling methods were identified using a cloning and sequencing based technique and included soil and environment-derived bacteria, as well as carcass or cadaver-derived organisms. This research demonstrated that pig (Sus scrofa ) carcass and human cadaver decomposition result in similar bacterial community changes in soil, confirming that pig carcasses are a good model for studying the microbiology of human decomposition. The inability to control for differences between donated human cadavers made understanding the human ii cadaver results difficult, whereas pig carcass study allowed many variables to be held constant while others were investigated. The information gained from this study about the bacteria associated with a cadaver and how the community alters over the course of decomposition may, in the future, enable the development of a forensic post mortem interval estimation tool based on these changes in the bacterial community over time. The findings in this thesis suggest that high variability between human bodies and their microflora is likely to present a challenge to the development of such a tool, but further study with emerging high-throughput molecular tools may enable identification of microbial biomarkers for this purpose. iii Acknowledgments I’d like to express my sincere thanks to all the wonderful people who have helped me get through my PhD. It has been immensely enjoyable and I will actually be sad (on some level) to finish. Others perhaps didn’t enjoy it as much as I did…… I’d like to thank my brilliant supervisor Dr Jacqui Horswell for guiding me through this project, kick-starting me when I needed it and telling me to go home when it was beginning to seem like I lived in the lab. Thanks too to my University supervisor Dr Geoff Chambers for giving me the freedom to diverge into interesting areas and for generally keeping things on track. A huge thankyou has to go to my US based supervisor Dr Arpad Vass for allowing me to stink out his lab and office for three months and for sampling with me at the Body Farm (not the nicest of jobs!!). Thankyou so much Arpad and Vicky, for taking such good care of me during my stay and making me feel like one of the family. Thanks to the kind staff at ORNL and the University of Tennessee’s Anthropology department for allowing me access to the lab and Body Farm. Thanks also to the others in the ESR lab: Tom, Andrew, Jen, Catriona, Danny, Penny, Mel, Justine, Steph, Alice and Cliff for not complaining TOO much about the smell of my samples and helping out when my sampling regime got a bit out of hand. Thanks to Phil and Marilyn for teaching, and fixing and re-running and explaining various things over and over! Also thanks to Justine the cloning guru and Steph the general everything guru for their endless patience with my questions. We really miss you Steph and Cat! Thanks also to Jeremy, who successfully decrypted science-geek speak into computer-geek speak and wrote super computer programmes, which made my life so much easier. iv I’d also like to thank my extended family for their support and ongoing interest in my weird choice of study topic, and my parents for helping with the pig cage building and sewing. Thanks must go to my 10 little pigs for the ultimate sacrifice, and to the generous people of the US who donate their bodies to science. I’ll never know the names in life of the bodies that were used in this research, but I respect and thank each of you for your gift. To my amazing Reid - Thankyou for being so interested in my research, for encouraging and inspiring me through the tough times and for promising to spoil me rotten once it’s all done! Finally….thankyou to the lab fairy for visiting the others for a while and letting me finally finish my lab work…… Dedication This thesis is dedicated to my grandfathers: Walter Chester and Murray Parkinson, who both, in their own ways, encouraged me to follow my heart……even if my heart took me in a very strange direction indeed!! I hope that I have made them both proud. v Table of Contents Abstract ............................................................................................................................ii Acknowledgments........................................................................................................... iv Dedication......................................................................................................................... v List of Figures ................................................................................................................. xi List of Tables................................................................................................................. xiv Abbreviations................................................................................................................ xvi Chapter 1: Introduction and Aims............................................................1 1.1 Aims ...................................................................................................................... 3 1.2 Research Approach ............................................................................................. 3 Chapter 2: Background and Literature Review......................................6 2.1 Human Decomposition........................................................................................ 6 2.1.1 Physical and Chemical Processes of Decomposition ....................................6 2.1.2 Variables Affecting Decomposition..............................................................8 2.1.3 Decomposition Microbiology......................................................................11 2.1.4 Soil Microbial Communities .......................................................................16 2.2 Post Mortem Interval Estimation .................................................................... 19 2.3 Molecular Analysis of Microbial Communities .............................................. 23 2.3.1 Genomic Microbial Community Analysis...................................................24 Chapter 3: General Methods ...................................................................33 3.1 Sample Characterisation................................................................................... 33 3.1.1 Sample Collection .......................................................................................33 3.1.2 Sample Preparation......................................................................................33 3.1.3 Sand sterility test .........................................................................................34 3.1.4 Buffering Capacity of Soils.........................................................................34 3.2 Biochemical Analyses ........................................................................................ 35 3.2.1 pH ................................................................................................................35 3.2.2 KCl Extractions ...........................................................................................35 + 3.2.3 Ammonium Concentration (NH 4 -N)..........................................................35 - 3.2.4 Nitrate Concentration (NO 3 - N) .................................................................36 3.3 Enzyme Assays................................................................................................... 37 3.3.1 Urease (EC 3.5.1.5) .....................................................................................37 vi 3.3.2 Fluorometric Assay .....................................................................................37 3.3.3 Microbial biomass .......................................................................................40 3.4 Molecular Biology Methods.............................................................................. 41 3.4.1 DNA Extraction...........................................................................................41