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Surfactants are a group of amphiphilic chemical compounds (i.e. having both hydrophobic and hydrophilic domains) that form an indispensable component in almost every sector of modern industry. Their significance is evidenced from the enormous diversity of applications they are used in, ranging from food and beverage, agriculture, public health, healthcare/medicine, textiles, oil & gas, and bioremediation. This PhD aimed to investigate two hydrocarbon-degrading bacterial strains, Halomonas sp. stain TGOS-10 and Pseudomonas sp. strain MCTG214(3b1), for their ability to produce biosurfactants and their usefulness for oil spill response. For this, three strategies were developed. First, the two strains were screened for production of surface-active compounds using sustainable substrates such as glucose and sunflower oil. Surface-active compounds were extracted, purified and their chemical structure was characterised with carbohydrate and amino acid assays, and NMR. Results revealed that both stains produced surface-active compound, TGOS-10 strain produced both an emulsifier and surfactant when grown on different substrates, whereas MCTG214(3b1) strain produced only surfactant. Second, the extracted and purified surfactants were tested for dispersion effectiveness at different concentrations and three oil types by utilising a standard baffled flask test. Both biosurfactants dispersed the crude oil at varying efficiencies depending on concentration and oil type but generally TGOS-10 showed better dispersing results than MCTG214(3b1). Lastly, in a case study from the northeast Atlantic, Illumina MiSeq sequencing was used to determine the response of the natural microbial community when exposed to either chemically-dispersed crude oil (commercial dispersant Finasol) or biosurfactant-dispersed oil (rhamnolipid from P. aeruginosa). In addition, parallel microcosms to determine hydrocarbon degradation were performed and analysed with Gas-Chromatography coupled with Flame Ionization Detection (GC-FID). During incubation for 4 weeks in roller-bottle microcosms, members of psychrophilic oil-degrading Colwellia and Oleispira initially dominated the microbial community in both the rhamnolipid and Finasol treatments. Thereafter, the community structure of these treatments significantly delineated. The microbial diversity was significantly greater in the treatment amended with rhamnolipid compared to that in the dispersant-amended treatment. GC-FID/MS analysis revealed that oil biodegradation was markedly enhanced in the Finasol-amended treatment. However, the "better-performing" qualities of the chemical dispersant Finasol may be in part, at least, conferred by other components that constituent its formulation, and biosurfactants, such as rhamnolipid, could potentially be developed into dispersant formulations with much improved qualities. Ecological null models were also used to better understand and quantify the relative importance of ecological processes in the assembly of microbial communities.

Hydrocarbon-degrading bacteria play a crucial role in the recovery of marine systems in the event of an oil spill, and associate with various species of eukaryotic phytoplankton in the ocean. There is a paucity of knowledge to explain the relationship between these types of bacteria living associated with microalgae, and their collective response to oil spills in future ocean acidification (OA) conditions has not been studied to date. This thesis presents the first investigation that aims to understand the response of these organisms under future atmospheric CO2 concentration (750ppm) and crude oil spills. Research was conducted on laboratory cultures of Emiliania huxleyi and natural community assemblage of subarctic surface seawater. Using high-throughput analysis of 16S rRNA sequencing, previously described key hydrocarbon degrading bacteria such as Marinobacter, Alcanivorax, and Oleispira were detected associated with microalgae. The response of bacterial community varied from being 1) negatively affected by OA and oil enrichment, 2) negatively affected by OA, but positively affected by oil enrichment under projected ocean acidification conditions, or 3) positively affected by OA and oil enrichment. Marinobacter and Methylobacterium were negatively affected by OA, which exacerbated the response of E. huxleyi to crude oil exposure. However, biodegradation of crude oil was not significantly affected. Marinobacter was also negatively impacted in the natural microbial community samples. Polaribacter was negatively affected in ocean acidification conditions and when exposed to crude oil enrichment. Colwellia was negatively impacted by OA but thrived during exposure to both crude oil and future ocean acidification conditions. Sulfitobacter and Psychrobacter was positively impacted by OA and oil pollution. Despite detection of hydrocarbonoclastic bacteria in natural community assemblage, their relative abundance in the bacterial community did not increase as expected after oil enrichment. Furthermore, no biodegradation of crude oil was detected in microcosms with natural microbial community. Highly abundant taxa in both spring and fall communities from the northeast Atlantic, such as Colwellia and members of families Rhodobacteraceae and Halomonadaceae, and the class Gammaproteobacteria, were persistent even when exposed to both stressors. In the event of an oil spill by the end of the century, OA favours selection of persistent and resilient bacteria that will outcompete hydrocarbonoclastic bacteria, thus delaying biodegradation and recovery from crude oil pollution in a future ocean.

Treating municipal wastewater is necessary to limit the impact carbonaceous, nitrogenous and phosphorus matter present in spent water may have on receiving aquatic systems. Conventional wastewater treatment systems employing the activated sludge or biological nutrient removal process as the main phase of treatment, demonstrate a high proficiency at removing these contaminants. Despite this, these processes are described as problem shifting, simply causing secondary pollution because of high energy consumed, production of waste sludge and greenhouse gases. To improve the environmental impact of wastewater treatment, particularly in light of stricter effluent discharge standards, treatment processes that have low energy consumption without affecting performance are needed. A potential, more sustainable biological treatment process to remediate the contaminants from wastewater is by using microalgae. Although this concept has been extensively researched, limited commercial development has been achieved. A major hindrance to the implementation of microalgae to treat wastewater is the cultivation process, which is one of the main cost and energy burdens, and as such would not result in the much-desired reduction in overall energy consumption of wastewater treatment. This thesis evaluated the performance of a microalgae treatment process for primary settled municipal wastewater (PSW) in a laboratory setting under static culturing conditions, to examine the feasibility of a low energy treatment process. Initial experiments assessed three freshwater microalga to treat PSW under both optimal (aerated) and static (non-aerated) culture conditions. From these results, Chlorella vulgaris identified itself as the most promising species, exhibiting high inorganic nitrogen and phosphorus removal. The availability of a suitable carbon substrate was determined to be the main limiting-factor affecting the algal treatment performance under static cultivation. To investigate this, initial experiments of PSW enriched with glucose (< 300 mg L−1) as an organic carbon source to facilitate the bioremediation by C. vulgaris was performed. Characterisation of the wastewater revealed significant reductions in NH3-N (from 28.9 to 0.1 mg L−1) and PO4-P (from 3.2 to 0.1 mg L−1) in just 2 days. Additionally, the exogenous glucose appeared completely removed from the wastewater after the first day. These achieved levels of treatment in respect of both the NH3-N and PO4-P were much higher than those recorded without C. vulgaris treatment with or without glucose enrichment. The reliability of this process was evaluated across a further three independent batches of PSW with varying compositions and organic carbon sources. The efficiency of the microalgae treatment process at reducing NH3-N and PO4-P was consistent in PSW enriched with organic carbon, resulting in > 90% reduction of the inorganic compounds in each batch. Lastly, to overcome the material cost of applying commercial sources of organic carbon, experiments were conducted to evaluate the use of the carbohydrate rich by-product, pot ale, from the production of malt whiskey as a carbon substrate to promote microalgae growth and remediation in PSW. In batch experiments, repeated three times with wastewater collected and treated separately and sequentially, the efficiency of the microalgae in pot ale enriched PSW demonstrated a high variability at reducing NH3-N and PO4-P, between 99 to 58% and 94 to 58% respectively. When operated under semi-continuous mode the microalgae demonstrated to be reliable in treating pot ale enriched PSW however, the removal efficiency in NH3-N, PO4-P and COD declined slightly in each subsequent cycle following the replenishment of PSW. The results of the pot ale enriched experiments highlight future research needs, such as the optimisation of nutrient ratios in the PSW and control over pH, to ensure a consistent and reliable treatment performance. Overall the application of C. vulgaris to treat enriched PSW, without aeration, offers a key area to develop as an alternative low energy, biological wastewater treatment option.

Omega-3 fatty acids, in particular docosahexaenoic acid (DHA), have been extensively studied for many decades for their health benefitting properties in pre-natal development, cardiovascular and Alzheimer diseases and enhancing the inflammatory immune response system. DHA has also been shown to be essential for the optimal development of fish, and therefore is an important ingredient in fish feed. However, due to fish oil and fish meal supplies currently facing many challenges (e.g. heavy metal contamination, environmental impacts, etc.), the demand for alternative sources of omega-3 fatty acids (FA) is predicted to rise in the near future. To meet these challenges, oleaginous microorganisms that produce omega-3 FAs have been explored as a potential new resource, with a particular emphasis on the thraustochytrid group. In this study, ten new strains of thraustochytrids, that were originally isolated from Scottish marine waters, were investigated for their biotechnological potential. The first phase of the project identified the new strains as a novel genus of thraustochytrid, for which the name Caledonichytrium matryoshkum gen. nov., sp. nov., is proposed. The description was based on a polyphasic analysis that employed phylogenetic analysis, biochemical signatures (PUFA and carotenoid profiles) and morphological and life cycle assessment studies. After identification, the strains were screened for their potential as DHA single-cell oil producers. The strains were assessed in two media types and at two time points of growth phase. With a view to their industrial application, a mathematical study was also included to seek opportunity for recycling by-product oil as biodiesel. The results showed that one of the strains, OL5TA, produced the highest relative level of DHA (63% of total FA) than any other strain reported to date in a screening study. However, the low final biomass concentrations reached (< 1 g L-1), and the low total lipid content measured (< 7% of dry cell weight, DCW), were considered major hurdles to overcome for industrial application. To address these issues, the optimisation of the culture conditions was carried out in the following stage. The results showed no consumption of glucose at 0.1% or 2% concentration, suggesting the inability of the strain to assimilate glucose. This may have hindered competitive biomass concentrations compared to that by other strains reported in the literature. To remediate this inability, a preliminary study was conducted to determine carbon source utilisation and carotenoid production to seek other routes for medium optimisation and biotechnological potential. The study concluded by identifying a potential route of exploitation for galactose and long carbon chain as sole carbon sources.

The Faroe-Shetland Channel (FSC) is an important gateway for dynamic water exchange between the North Atlantic Ocean and the Nordic Seas. In recent years it has also become a frontier for deep-water oil exploration and petroleum production, which has raised the risk of oil pollution to local ecosystems and adjacent waterways. In order to better understand the factors that influence the biodegradation of spilled petroleum, a prerequisite has been recognized to elucidate the complex dynamics of microbial communities and their relationships to their ecosystem. This research project was a pioneering attempt to investigate the FSC's microbial community composition, its response and potential to degrade crude oil hydrocarbons under the prevailing regional temperature conditions. Three strategies were used to investigate this. Firstly, high throughput sequencing and 16S rRNA gene-based community profiling techniques were utilized to explore the spatiotemporal patterns of the FSC bacterioplankton. Monitoring proceeded over a period of 2 years and interrogated the multiple water masses flowing through the region producing 2 contrasting water cores: Atlantic (surface) and Nordic (subsurface). Results revealed microbial profiles more distinguishable based on water cores (rather than individual water masses) and seasonal variability patterns within each core. Secondly, the response of the microbial communities to crude oil was investigated in laboratory-based microcosms. Microbial communities from all water masses exhibited hydrocarbon biodegradation activity at average FSC temperatures (4°C), albeit with markedly delayed and potentially slower response in comparison to those exposed to moderate control temperatures (20°C). A collection of bacterial isolates, comprising of 230 FSC strains with putative hydrocarbonoclastic activity was created, which included psychrotolerant members belonging to the genera Marinobacter, Pseudoalteromonas, Cycloclasticus, Halomonas, Thalassolituus and Glaciecola. Lastly, a sophisticated molecular technique called DNA-based stable isotope probing (DNA-SIP) was used to directly target and identify hydrocarbon-degrading taxa that may not be easily amenable to cultivation. Using DNA-SIP, hydrocarbonoclastic FSC strains affiliated with the genera Phaeobacter and Lentibacter were identified, along with strains affiliated with known hydrocarbon-degraders from the genera Thalassolituus, Alcanivorax, Oleispira, Glaciecola, Marinobacter and Cycloclasticus. Correlating the findings from all three experiments, revealed that ~41% of the baseline FSC microbial community constituted bacteria affiliated to genera with hydrocarbon-degrading capacities. Their response to the presence of hydrocarbons, however, appeared to be largely influenced by temperature. This work is the first to establish a microbial baseline for the FSC and to investigate the microbial repose to crude oil in the water column of the region. Results are expected to contribute to the development of biotechnologies and oil-spill mitigation strategies tailored for the FSC region in the event of an oil spill.

Crude oil spills damage marine ecosystems due to the potentially toxic nature of the petrochemical hydrocarbon constituents and their recalcitrance to degradation. Polycyclic aromatic hydrocarbon components (PAHs) are one group of hydrocarbons in crude oil that are of particular concern due to their genotoxicity and potential to bioaccumulate. Their potential to cause damage in marine environments can be mitigated by the presence and activities of hydrocarbonoclastic bacteria. The phycosphere of marine eukaryotic phytoplankton (micro-algae) has recently been shown as an important biotope where hydrocarbonoclastic bacteria can be found, and the association between these organisms is largely unexplored. This thesis presents new insight into the relationship between these organisms by performing enrichment experiments with crude oil and individual hydrocarbon substrates, as well as wholegenome analysis of an algal-associated hydrocarbon-degrader, and using molecular probes for the in situ visualization (by CARD-FISH) and whole-community analysis (by Flow-FISH) of hydrocarbonoclastic bacteria associated with laboratory cultures and field samples of micro-algae. Results demonstrated variations in the tolerance of different diatom species to PAHs, and that intermediate metabolites formed from the partial biodegradation of PAHs can be more harmful to diatoms compared to that of their parent PAH compounds. Thalassiosira pseudonana presence enhanced phenanthrene dissolution and PAH-degrading bacteria formed cell clusters in EPS aggregates. The genome of an obligate hydrocarbonoclastic bacterial species (Polycyclovorans algicola) found associated with marine micro-algae possessed genes involved in cell communication, horizontal gene transfer and nutrient sharing that may play an important role in the organism’s association with its eukaryotic host cells. Indeed, these interactions are likely to allow these hydrocarbonoclastic bacteria to be supported on the phycosphere of micro-algae in the absence of petrochemical exposure. Microcosm experiments with field samples of micro-algae and bacterial consortia showed that crude oil biodegradation, in particular the PAH fraction, was enhanced compared to that by the free-living bacterial community, and revealed certain groups (e.g. members of the order BD7-3) that had not previously been reported to become enriched in the presence of crude oil. Using Flow-FISH, epibiotic members of the genus Marinobacter were found associated with micro-algae in sea surface field samples, and CARD-FISH was used to show this same group associated with the soft tissues of the coral Lophelia pertusa from polyp samples collected from different coral mounds in the deep North Atlantic.