University of Huddersfield Repository Kyeremeh, Isaac Ampaabeng Characterisation of Novel Isosaccharinic Acid Degrading Bacteria and Communities Original Citation Kyeremeh, Isaac Ampaabeng (2018) Characterisation of Novel Isosaccharinic Acid Degrading Bacteria and Communities. Doctoral thesis, University of Huddersfield. This version is available at http://eprints.hud.ac.uk/id/eprint/34509/ The University Repository is a digital collection of the research output of the University, available on Open Access. Copyright and Moral Rights for the items on this site are retained by the individual author and/or other copyright owners. 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For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected]. http://eprints.hud.ac.uk/ Characterisation of Novel Isosaccharinic Acid Degrading Bacteria and Communities Isaac Ampaabeng Kyeremeh, MSc (Hons) A thesis submitted to the University of Huddersfield in partial fulfilment of the requirements for the degree of Doctor of Philosophy Department of Biological Sciences September 2017 i Acknowledgement Firstly, I would like to thank Almighty God for His countenance and grace all these years. ‘I could do all things through Christ who strengthens me’ (Philippians 4:1) Secondly, my heartfelt gratitude and appreciation go to my main supervisor Professor Paul N. Humphreys who gave me the opportunity to work with him and provided all the necessary support up to the end of my research. Prof., your constant suggestions, constructive criticisms and constant reminders have played a pivotal role to the success of this research and I will forever be grateful. Also to my second supervisor, Professor Andy Laws for your time and the opportunity for mr to work in your analytical laboratory. My next thanks go to my dear wife, Mrs Faustina A. Kyeremeh who has been with me through all these years. All I can say is: God bless you; for words cannot explain the immense support you have given to me. Having to put up with constant absences, stress and all the hardships, I say thank you. To the rest of the family, my children, Mr and Mrs Tetteh as well as Mr and Mrs Sateklah for your special support. Finally, thanks to you all my colleagues in the research group and also in the department especially, Dr Simon P. Rout, Dr Sohaib Sadique, Dr Jack Blackburn, Mr Zohier Salah, Mustapha Sulleyman, Hansel Ngo, Ms Makhosi Mgombane and all who contribute in diverse ways to make my research at the University of Hudderfield a success. ii Dedication I would like to dedicate this work to two people. Firstly, to my dad of blessed memory, Nana Appiah Kubi for all the investments he made towards my education but sadly is not here to see its success. Secondly, to my lovely, two-year old daughter Lizianna Sheridan A. Kyeremeh who refused to go to bed but did all night long wake keeping with me during the preparation of the thesis. iii Abstract The current plan for the permanent disposal of Low Level and Intermediate Level nuclear wastes is the cement-based geological disposal where the wastes will be disposed off in geological disposal facility (GDF). The chemical evolution of the GDF is expected to cause cellulosic materials in the waste to degrade into cellulose degradation products (CDP) of which isosaccharinic acids (ISA) are the major components. ISAs are reported to form complexation reactions with radionuclides, potentially enhancing their migration out of the GDF. Recent studies have shown that microbial consortia indigenous to anthropogenic sites can potentially degrade these ISAs but have focused on the use of different nitrogen sources Ca(ISA)2 as analogue for CDP. This study therefore aimed at characterizing novel ISA degrading bacteria and investigate the metabolic potentials of microorganism within soil sediments from Harpur Hill site to biodegrade ISAs and assess the impact of + + using Ca(ISA)2 or CDP with NH4 against NH4 free systems on the chemical and microbial community evolution under conditions representative of the GDF. In a batch/fed microcosms, microorganisms within the soil sediment were able to biodegrade ISAs in pH ranging from 9-11.5 irrespective of the source. The microbial community evolution in these systems were however different suggesting that the type of carbon source and the presence of a nitrogen source impacted on the selection of these communities. The evolution of the microcosms gave rise to a complex methanogenic, polymicrobial communities where the degradation of ISAs led to the formation of acetic acid and gases including CH4, CO2 and H2. The formation of these gaseous products are likely to contribute to the pressurization of the GDF as a result of which the porosity and permeability factors should be taken into account in the formulation of the cement backfill materials. Molecular characterization of the ISA degrading communities and pure isolates (Exiguobacterium sp. strain Hud and Oceanobacillu sp. strain Hud) will allow for studies into genes associated with ISA degradation which is currently lacking in the literature. The Harpur Hill site presents a diverse pool of microorganisms with the metabolic potentials to degrade ISA hence it could be a good candidate site for the GDF. iv Contents Introduction ....................................................................................................... 1 The United Kingdom’s Nuclear Waste Legacy .......................................... 2 Categories of nuclear wastes ................................................................. 2 The scope of nuclear wastes in the UK ................................................ 4 Long-term management plan for LLW and ILW in the UK ...................... 5 The Generic Safety Functions of the multi-barrier system ................... 7 The Cement-based disposal concept for LLW/ILW ............................. 8 Structure of Cellulose and the Degradation of Cellulosic Materials Relevant to the Cement-based disposal concept of ILW ................................. 11 Radiolytic Degradation of Cellulose .................................................. 12 Anaerobic Alkaline Degradation of Cellulose .................................... 13 Microbial degradation of cellulose ..................................................... 16 Prevalence of ISA and other CDPs in the natural Environment ............... 17 Evidence of microbial degradation of ISA under repository conditions ....................................................................................................................... 18 Methods for the identification and characterisation of microbial populations within an environmental sample .................................................. 26 Culture-based techniques .................................................................... 26 Microcosm investigations ................................................................... 27 Biochemical/Physiological characterisation ....................................... 29 Biochemical markers Based methods ................................................. 30 Molecular based methods of microbial community analysis ............. 30 Partial community Analysis ................................................................ 31 v Microbial lipid analysis ...................................................................... 34 Whole community analysis ................................................................. 37 Overview of sampling sites analogous to the GDF for microbial investigations .................................................................................................... 41 Biochemical processes relevant to anaerobic microbial degradation of carbohydrate substrates .................................................................................... 44 Fermentation ....................................................................................... 44 Acetogenesis ....................................................................................... 46 Syntrophism ........................................................................................ 48 Methanogenesis ................................................................................... 49 Aims and Objectives ....................................................................................... 52 Materials and Methods .................................................................................... 54 General Reagents ...................................................................................... 55 Growth media Preparation .................................................................. 55 Mineral media ..................................................................................... 55 Preparation and characterization of Cellulose Degradation Products (CDP) .......................................................................................................................... 57 Preparation and characterization of Calcium alpha-Isosaccharinic Acid (Ca[(α-ISA)2]) .................................................................................................