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This thesis described the development of DNA Stable Isotope Probing (SIP) with the goal of identifying microorganisms and genes involved in the metabolism of aromatic compounds. The research had three main components. Results have been presented in chapters 3, 4 and 5. • Chapter 3 focused on investigating the effect of growth on 2H labelled compounds, developing a method for the isolation of 2H labelled DNA and understanding the limitations of this approach using a range of pure cultures. • Chapter 4 describes experiments used to validate the 2H DNA SIP method using environmental samples and compare SIP results aiming to identify 16s rRNA and dioxygenase sequences using 2H and I3C labelled benzoic acid. • Chapter 5 utilised the methods developed in chapters 3 and 4 to investigate the organisms and genes involved in the degradation of naphthalene vs. methylnaphthalenes in a mixed enrichment culture inoculated with P AB contaminated soil from a former Belfast gas works site. The aim of these experiments was to demonstrate that 2H DNA SIP could be used to gain new information on methylated P AB microbiology and be used specifically for the isolation of biocatalytic genes. • Chapter 1 Presents an introduction to this research • Chapter 2 describes the materials and methods used during the course of this research. • Chapter 6 summarises the work as a whole, suggests future work and discusses the implications of this research in the wider scientific community. • Chapter 7 References

Arene cis-dihydrodiols are compounds produced exclusively during the bacterial oxidation of aromatic substrates through the action of highly stereo- and regio-specific dioxygenase enzymes. cis-Dihydrodiols are important starting materials in the synthesis of valuable stereoisomeric molecules, which in recent years have attracted the attention of research groups in industry and academia. . The major objective of the present thesis was to investigate the formation and functionalisation of cis-dihydrodiols using chemo- and bio-catalytic methods. Oxidation and reduction reactions of cis-dihydrodiols constitute a common'factor across the project since they lead to the formation of the corresponding catechols and the partially (cyclohexene-) or . fUlly hydrogenated (cyclohexane-) cis-diols. Special attention was paid to methodological developments, the type of catalyst used and the comparison between different synthetic procedures in terms of efficiency, selectivity, sustainability and potential scalability. In general, the resulting products are valuable precursors with application in a variety of industrial processes. Two methods are presented for the production of 3-substitued catechols. In both cases the first synthetic stage involved the bio-catalyzed oxidation of a monosubtituted benzene to the corresponding cis-dihydrodiol, using a toluene dioxygenase enzyme present in Pseudomonas putida UV4 or in Escherichi.a coli DH5(pCLtdo). SUbsequently, two different dehydrogenation routes were taken to convert the cis-dihydrodiol to the corresponding catechol, using either a heterogeneous catalyst (Pd/AC) or a cis-diol dehydrogenase enzyme (NDDR or BDD) present in a recombinant E. coli strain. The performance and potential capability of NDDR and BDD for other dehydrogenation reactions were investigated, including kinetic resolution and catalytic asymmetric synthesis of enantiopure arene cis-dihydrodiols, catechols, ketoalcohols and alkene cis-diols. The synthesized catechols were used as starting compounds for reactions involving the hydroxyl and the substituent groups, as well as ringcleavage reactions. Furthermore, a mild and reliable procedure for the partial and total hydrogenation of 3-substituted benzene cis-dihydrodiols using Rh/G was developed.

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