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Isolation and Characterization of ISA Degrading Alkaliphilic Bacteria

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Isolation and Characterization of ISA Degrading Alkaliphilic Bacteria Isolation and Characterization of ISA Degrading Alkaliphilic Bacteria Zohier Salah (Researcher) A thesis submitted to the University of Huddersfield in the partial fulfilment of the requirements for the degree of Doctor of Philosophy School of Applied Science December 2017 Acknowledgment Praise be to Allah through whose mercy (and favors) all good things are accomplished Firstly, I would like to express my sincere gratitude to my main supervisor Professor Paul N. Humphreys who gave me the opportunity to work with him and for the continuous support of my PhD study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. Also, I wish to express my appreciation to my second supervisor, Professor Andy Laws for all the assistance he offered. Without they precious support it would not be possible to conduct this research. Special thanks go to the Government of Libya for providing me with the financial support for this study. I would also like to thank all the laboratory support staff in the School of Applied Sciences, University of Huddersfield, for all the support. Sincerely appreciations also go to my family especially, my parents and my virtuous wife Zainab, my son Mohamed and the extended my family, especially my brother Mr. Ahmed Salah. Finally, but by no means least, thanks to all my colleagues in the research group who helped with experience, ideas and discussions during my studies, Dr Simon P Rout, Dr Isaac A Kyeremeh, Dr Christopher J Charles and to all who contributed in diverse ways to make my research at the University of Huddersfield a success. 2 Dedication I wish to dedicate this piece of work to my wonderful wife, Zainab and my son Mohammed who have spent all the time with me throughout my study in the England. Abstract Radioactive waste disposal in the UK is expected to be managed via the construction of a deep geological disposal facility (GDF) backfilled with cementitious grouts. Post closure the repository is expected to have a hyper alkaline, anoxic environment. The UK’s intermediate- level radioactive waste (ILW) inventory contains significant quantities of cellulosic materials that expected to undergo alkaline hydrolysis under repository conditions to form Iso-saccharinic acids (ISA). The isomers of ISA (α- and β-) are able to form water-soluble complexes with radioelements, increasing their solubility and enhancing their migration. The biological removal of ISA through microbial activity would therefore have a positive impact on repository performance by reducing complex formation. This research aimed to isolate pure cultures capable of degrade ISAs under anaerobic and alkaline conditions analogous to GDF. The microcosms of mineral media and CDPs-fed cycle as a sole carbon source inoculated by alkaline soil samples from the Buxton site. The degradation process monitored under a fermentation and an anaerobic respiration by adding terminal electron acceptors. All processes carried out under alkaline and strict anaerobic conditions. Different types of agar plate media used to obtain pure culture. The results of the current study indicate that alkaliphilic bacterial communities were capable of fermenting ISA to acetate up to pH 11.0. In addition, the ISA (α- and β- isomers) degradation through terminal electron acceptors at pHs (7.0, 8.0, 9.0 and 10.0) were tested, resulted to a significant amount of ISA was degraded in Nitrate-reducing culture, and small amount in Iron (III)-reducing culture, whilst there was no sign of degradation in Sulphate-reducing culture. A 16SrRNA gene sequencing showed a significant reduction of the bacterial community in the microcosms compared with the crude soil sample. The phyla that detected in all microcosms at different pH values dominated by Firmicutes, followed by Proteobacteria, Bacteroidetes, Actinobacteria and Archaea Euryarchaeota. However, in pure culture many of the bacteria isolated were unable to degrade ISA eventhough they had been isolated and enriched under ISA degrading conditions. Only two bacterial strains purified which were capable of degrading ISA. These strains Macellibacteroides fermentans HH-ZS a Gram negative, strictly anaerobic bacterium and a strain of Aeromonas sp. The biochemical analysis, metal and NaCl tolerance, pH profile, biofilm and extracellular polymeric substances detection, fatty acid methyl ester profile, and whole genome sequencing analysis used for characterization of the some isolated Alkaliphiles. The phylogenetic and biomarker results led to identifying a novel strain of Macellibacteroides fermentans HH-ZS strain is the first Gram negative, strictly anaerobic bacteria able to degrade ISA. Future work; the genome of the M. fermentans HH-ZS strain harboured a number of carbohydrate degrading enzymes, which merit further investigation to determine the metabolic pathways associated with ISA degradation. In addition, some of isolated Alkaliphiles considered as rare strains and some of them identified as new strains; further investigation to find the possibility to introduce these isolates in the bioremediation and an industrial application. 1. Contents Chapter 1 1 1. Introduction ............................................................................................................................ 1 The Fate of Cellulosic Materials .................................................................................... 4 Chapter 2 19 2. Materials and methods .......................................................................................................... 19 Commercial media ....................................................................................................... 23 Prepared medium .......................................................................................................... 23 Ultra Clean® Microbial DNA Isolation Kit .................................................................. 24 Isolation genomic DNA from soil sample .................................................................... 24 Griffiths method for DNA extraction ........................................................................... 24 v Quantification of DNA sample..................................................................................... 24 DNA Electrophoresis ................................................................................................... 24 Amplification of 16S rRNA gene by PCR ................................................................... 24 PCR product purification ............................................................................................. 25 Analysis of bacteria sequencing ................................................................................... 25 Construction of phylogenetic trees ........................................................................ 25 Quantification of Nitrate, Sulphate and Iron concentrations ........................................ 35 Chapter 3 38 3. Results and discussion .......................................................................................................... 38 vi Key findings ................................................................................................................. 89 pH profiles .................................................................................................................. 140 Discussion ........................................................................................................... 142 Biochemical characterization of alkaliphilic isolates ................................................. 143 Heavy metal and sodium chloride tolerance .............................................................. 147 Discussion ........................................................................................................... 148 Production of extracellular polymeric substances and biofilm formation .................. 149 Discussion .......................................................................................................... 151 Microscopic characterisation of spore-forming alkaliphilic isolates .......................... 153 Phylogenetic analysis of the spore forming bacterial isolates .................................... 155 Biochemical characteristics and pH profiles of the bacterial isolates ........................ 157 Key findings ............................................................................................................... 163 Characterization of Aeromonas sp. ZS strain (BI 55) ................................................ 165 Phylogenetic analysis of Aeromonas sp. ZS strain (BI 55) ................................. 165 Morphological and metabolic characterisation of Aeromonas sp. ZS (BI 55) .... 167 vii Key findings ........................................................................................................ 170 Draft Whole Genome Sequence of the Cl. tertium ZS strain (BI85) ......................... 170 Characterisation and WGS of Alishewanella sp. HH-ZS strain ................................. 172 Phylogenetic analysis of Alishewanella aestuarii HH-ZS strain (BI28) ............. 172 Morphological and metabolic characterisation of Alishewanella sp. HH-ZS strain ............................................................................................................................................. 174 Draft Whole Genome Sequence .......................................................................... 179 Key findings .......................................................................................................
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