Ph. D. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.

Les écosystèmes côtiers sont des milieux complexes au sein desquels les communautés microbiennes, jouant un rôle majeur dans leur fonctionnement et leur maintien, s’adaptent et sont tolérantes à des conditions environnementales fluctuantes. En effet, au rythme des marées et de l'activité de la macrofaune, des oscillations oxie/anoxie influencent la composition et la dynamique des communautés microbiennes et par conséquent leur implication métabolique. Afin d’appréhender le devenir du pétrole dans ces écosystèmes, il est donc indispensable d’apporter des connaissances sur l’écologie des microorganismes intervenant dans son élimination, notamment dans des conditions oscillantes anoxie/oxie. Ainsi, ce travail de thèse a eu pour objectif de décrypter l’assemblage de communautés microbiennes hydrocarbonoclastesde sédiments intertidaux soumises à des oscillations anoxie/oxie en présence de pétrole lors d’une expérience en bioréacteurs. Les réponses écologiques des communautés bactériennes globales et de micro-organismes sulfato-réducteurs en conditions oscillantes ont pu être décrites en comparaison avec celles obtenues en conditions d’oxie ou d’anoxie permanentes, par l’analyse des données obtenues par séquençage haut-débit des gènes de l’ARN 16S et dsrB au niveau transcriptionnel. Ces études comparatives ont mis en évidence des profils écologiques en réponseaux conditions oscillantes, pouvant être répandus dans différents environnements marins côtiers. En réponse à ces conditions particulières, de nombreux microorganismes semblent avoir le potentiel à tolérer et/ou s’adapter aux différentes conditions d'oxygénation. Cette capacité d’acclimatation rapide des communautés bactériennes aux conditions oscillantes se sont accompagnées de capacités de dégradation équivalentes ou supérieures dans ces conditions par rapport à la condition d’oxie permanente montrant l’influence des oscillations anoxie/oxie sur le devenir du polluant dans les environnements pollués soumis à ces conditions. / Coastal ecosystems are complex environments in which microbial communities, playing a major role in their functioning and maintain, are tolerant and adapt to changing environmental conditions. Indeed, the tides and the macrofauna’s activity generate oxic/anoxic oscillations which influence the composition and dynamics of microbial communities and consequently their metabolic in volvement. To understand the fate of oil in these ecosystems, it is essential to provide knowledge on the ecology of microorganisms involved in these systems, taking into account anoxic/oxicoscillating conditions. Thus, this thesis aimed to decipher the organization of hydrocarbonoclastic microbial communities inhabiting intertidal sediments, when they are subjected to anoxic/oxic oscillations in an experiment in bioreactors with oil addition. Ecological responses of bacterial communities and sulfate-reducing microorganisms in oscillating conditions have been described comparing with those obtained with permanent oxic or anoxic conditions, using high-throughputsequencing analyses of the 16S rRNA and dsrB genes at the transcriptional level. These comparatives studies have highlighted ecological profiles in response to the oscillating conditions, which can be prevalent in different coastal marine environments. In response to these particular conditions, many organisms seem to have the potential to tolerate and / or adapt to the different conditions of oxygenation. This rapid acclimation capacity of bacterial communities tothese changing conditions have been accompanied by equivalent or greater degradation capacity under these conditions compared to the permanent oxic condition, showing the influence of the anoxic/oxic oscillations on the fate of pollutant in environments subjected tothese conditions.