Salah, Zohier (2018) Isolation and Characterization of ISA Degrading Alkaliphilic Bacteria. Doctoral thesis, University of Huddersfield.

Radioactive waste disposal in the UK is expected to be managed via the construction of a deep geological disposal facility (GDF) back filled 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 (7.0,8.0,9.0and 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 even though 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.

Salah THESIS.pdf - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.

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