The deleterious effects of maternal diatom diets on copepod embryonic development have been puzzling the scientific community for the past 15 years. Since the discovery of the first anti-mitotic compounds, polyunsaturated aldehydes deriving from fatty acid oxidation,our knowledge of the oxylipin metabolism in diatoms has been continuously increasing. Not only have new oxylipin compounds been identified, but it is becoming apparent thatoxylipin metabolism is highly complex and specific. This thesis contributes to the understanding of oxylipin metabolism in diatoms by studying the activity of diatom lipoxygenases, the enzymes responsible for the first oxidative step in transforming polyunsaturated fatty acids into oxylipins. Lipoxygenase activity was measured by a colorimetric and a polarographic assay, and its relationship to oxylipin production was examined. Lipoxygenase metabolism in diatoms was studied under natural conditions during a bloom at sea, under semi-natural conditions during a mesocosm experiment, and in the laboratory, with special emphasis on <i>Skeletanema marinai</i>. The effect of diatom oxylipin production on copepod reproductive success was also examined. Increased oxylipin production was found with the onset of the stationary phase in culture and during the decline of the mesocosm bloom, which indicates a regulatory role foroxy lipins in bloom demise, even though this could not be verified at sea. Lipoxygenase activity and oxylipin production were highly variable and specific to the level of clones. The most important conclusion to be drawn from the present experiments is the lack ofcorrelation between lipoxygenase activity and oxylipin production. This strongly indicates that other compounds still need to be identitied and that we do not yet have a complete picture of oxylipin metabolism in diatoms.

The planktonic diatom <i>Pseudo-nitzschia multistriata</i> appeared in plankton samples taken at the MareChiara station in the Gulf of Naples in 1995 and has appeared ever since in autumn, and since 2004 also in summer. This thesis focuses on population genetic structure of the species using the Internal Transcribed Spacers in the rDNA as well as seven polymorphic microsatellites. Cells isolated from plankton samples taken in 2008 and 2009 were grown into strains from which ITS-sequences and microsatellite fingerprints were gathered. Microsatellite screening uncovered two populations in sympatry in 2008, only one of which reappeared in 2009. The ITS sequences of the strains were of distinct types,called A-type, B-type and a mixed type (NB). The two populations also showed distinct ITS-type proportions, supporting their distinctness. Intra-population proportions of ITS-types among strains were similar in summer and autumn 2009,indicating stability over the growth season. But proportions differed between 2008 and 2009, suggesting substantial immigration the second year. These results suggest that, <i>P. multistriata</i> in the Mediterranean consists of a dynamic patchwork of populations. Microsatellite inheritance was studied over the vegetative and sexual parts of the biennial life cycle of <i>P. multistriata</i>. Substitutions were recorded in strains rescreened after several months of maintenance. Longer microsatellites changed more frequently than shorter ones and di-nucleotide ones more than tri-nucleotide ones. Strains interbred independently from population assignment. Comparisons of parental and offspring genotypes showed microsatellite inheritance patterns generally following Mendelian rules. Genetic identity of sister cells emerging from zygotes formed on the same parental gametangia indicate that one of the two nuclei resulting from the first meiotic division is eliminated. Each maternal gamete fuses with a paternal gamete, resulting in two zygotes, which, in this case, were genotypically identical. Thus, mutations boost allele diversity and biennial sexual reproduction boosts ITS-genotype variability and genotypic diversity.

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Macroalgae represent a diverse and abundant resource, containing an array of unique chemicals with useful properties. These chemicals include: alginate, a long chain carbohydrate with gelling properties; laminarin, a carbohydrate consisting of glucose residues, which is readily fermented; mannitol a sugar alcohol that can be used as an artificial sweetener and fucoidan, a sulphated polysaccharide famed for its biomedical properties. Their current use in industry is minor, with the main focus being as a food source and for alginate extraction. However, there is great potential for this feedstock in chemical and fuel production, especially for biorefinery development, which makes use of the whole resource by providing multiple products from one feedstock. Brown macroalgae offer the most promising option in Northern Europe, being the largest and most fast growing of the seaweed species, as well as being plentiful around the coast of the UK with the potential for cultivation alongside harvesting from wild stock. A potential barrier to the use of seaweed in industry is their seasonal variation in chemical content. In order to fully understand this, a study detailing the variations in carbohydrates, protein and ash, as well as a detailed study into the variation in composition and structure of fucoidan, identified as the most valuable of the potential extraction products due to its interest in the pharmaceuticals market, have been conducted. Three species of brown macroalgae, Fucus serratus (FS), Fucus vesiculosus (FV) and Ascophyllum nodosum (AN), have been analysed over a 12 month period. The results indicate that mannitol, laminarin and fucoidan are all highest at the end of the growing season in late summer and that ash, alginate and protein are highest during the winter months. The composition and structure of fucoidan is also seen to vary over the year, with FS having the highest sulphate content and results indicating a consistently more branched structure than was seen for FV and AN. In order to make the best use of the macroalgal feedstock, a three step hydrothermal microwave assisted biorefinery is proposed, with utilisation of the waste as a feedstock for fuel production or as a fertiliser being considered. For this, a sample of FS, identified in the seasonal variation study to have the best potential for chemical extraction, has been used. A low temperature step at 50°C in water firstly removes mannitol and a portion of the salts, followed by processing at 120°C in water to extract fucoidan and alginate. Alginate is precipitated from the extract with calcium carbonate and fucoidan with ethanol. The final step is processed at 120°C with sodium chloride to extract the remaining alginate from the residue. A mass balance of the proposed biorefinery shows that 90% of mannitol, 79% of fucoidan and 79% of alginate have been extracted during processing. A study into the quality of the fucoidan extracted by microwave heating is comparable to that extracted from the raw biomass by conventional means. A comparison of microwave and conventional heating shows the benefits in using microwaves, with decreased extraction temperature and a full energy balance of the system significant energy reductions associated with microwave heating on a laboratory scale.

Phytoplankton in the ocean are a key component of the global carbon cycle and the base of most marine food webs. They contribute approximately one half of total global primary production, but are restricted to the euphotic zone, where sunlight is sufficient for photosynthesis. Due to ocean stratification nutrients become limiting in the near-surface ocean and the distribution of phytoplankton is influenced by the upward flux of nutrients from below. In situ measurements of primary production and nutrient fluxes are generally sparse and questions still remain about quantifying how the physical mechanisms supplying nutrients balance the amount of growth. Using a year long observational dataset from ocean gliders and mooring data this thesis investigates primary production in the North East Atlantic and the associated vertical nitrate fluxes into the euphotic zone. A method was developed to estimate primary production from glider chlorophyll fluorescence and photosynthetic active radiation data using a primary production algorithm designed for satellite data. Primary production in the subsurface was quantified from the model, showing the formation of a subsurface primary production maximum, which contributed 23% of the total annual primary production. Concurrent nitrate fluxes are calculated from mooring vertical velocities and diffusivity measurements using a density-nitrate relationship derived from cruise CTD bottle samples. An estimate of the convective flux was also estimated using glider mixed layer depths. An annual timeseries of sub-daily estimates of primary production and nitrate fluxes was therefore obtained. Annual budgets show that nitrate fluxes could support between 75 and 102% of the observed primary production. The vertical advective flux contributes the largest vertical flux to the nitrate supply, and is therefore of greater significance than previously considered. Notable small scale temporal variability was observed in both the timeseries of primary production and nitrate fluxes, demonstrating an important influence from submesoscale and mesoscale processes on phytoplankton growth.

Marine photosynthetic microalgae have great potential in biotechnology. They have huge genetic diversity and naturally make an array of metabolites that are precursors in high value products such as fuels and pharmaceuticals. They do not compete with agriculture for land or fresh water and can be used to reduce industrial carbon-emissions. In order to realize this potential however much work needs to be done to overcome the engineering challenges of growing microalgae on large scales and developing the genetic tools required to increase the yield and diversity of products synthesized by cells. Irrespective of the type of microalgal species selected for growth, the efficiency at which light energy is converted into product, the 'photosynthetic conversion efficiency' sets a fundamental limitation on the potential yield. In natural systems as much as 90% of absorbed light energy is re-emitted as heat or fluorescence, representing a major loss in overall efficiency. In this thesis a high-throughput pipeline using random mutagenesis and live single cell sorting has been used to isolate two cell-lines of the eukaryotic microalgae Dunaliella tertiolecta with reduced chlorophyll content (termed lca1 and lca2). As there is no published genome for D. tertiolecta and the species is difficult to transform, this approach represents a feasible method to develop improved cell-lines from any microalgal species. These cell lines are characterized physiologically and shown to increase the maximum rate of chlorophyll-normalized photosynthesis Pmax by 289 (lca1) and 131% (lca2) respectively. The molecular basis of these random mutations characterized by transcriptomics using next-generation sequencing approaches, helps define the differences in regulation in light-harvesting and photosynthesis between the lca1, lca2 and wild-type. The approaches applied in this thesis therefore show how microalgal strains with poor genetic characterization can rapidly be selected for biotechnological applications, and providing new gene targets and valuable insights into the fundamental mechanisms of photosynthesis.

No description available.

No description available.

The research presented here regards the biosynthetic origins of several secondary metabolites isolated from red algae of the genus Laurencia. This thesis has been separated into two major parts, and includes an appendix cataloguing the experimental procedures and spectroscopic data. Part I covers the epoxidation of bromoallenes, as inspired by the previously unknown biosynthetic link between two natural products isolated from Laurencia obtusa, one bearing a bromoallene and the other an α,β-unsaturated ester. Employing model bromoallene 1 and a range of epoxidation methods, a procedure was developed that would generate α,β-unsaturated acids 2 in low, but reproducible yields. Isotopic labelling of the substrate with deuterium and carbon-13 allowed elucidation of the mechanism, by observation of the migration of these atoms. Part II of this thesis reports the completion of the total synthesis of a range of Laurencia secondary metabolites. The initial targets of this project proved to be unobtainable, most likely due to the sensitivity of the allyl ether group under reductive conditions. An alternative route was successfully applied to bypass this problematic step, allow the synthesis of a range of Laurencia natural products and provide insights into their biosynthesis. Positive identification of these compounds was achieved by a Δδ analysis of the collected 1H & 13C NMR spectra, with that reported in literature.

Diatoms contribute about 50% of global primary production and are on of the most diverse phytoplankton groups. Additionally, they form the basis of most marine food webs and play an important role in elemental cycles such as carbon and silica. Global warming impacts the diversity and productivity of marine ecosystems as temperature is considered a strong selecting agent underpinning global diversity patterns of marine phytoplankton. In order to gain insights into diatom distribution and diversity in the Atlantic Ocean, we analysed 18S rDNA ribotypes over a broad spatial scale from the Fram Strait to the South Atlantic. Diversity patterns were related to environmental metadata in order to identify main drivers. Our results indicate that salinity had a negative effect on diatom diversity in the Fram Strait transect with stations showing low diversity at high salinities. In contrast, diatom diversity in the Atlantic Ocean was negatively correlated to temperature with high temperature showing low diatom diversity. The order of Coscinodiscales showed a, formerly unknown, cosmopolitan distribution and was the overall most abundant species. With this study we provided an updated estimate of diatom distribution and diversity in the Atlantic Ocean. Phytoplankton physiology is highly temperature dependent and despite the importance of temperature as a major driver of marine phytoplankton evolution, the molecular mechanisms of adaptive evolution under temperature selection are largely unknown but instrumental for predicting how marine phytoplankton will respond to a changing ocean. Here we provide evidence, based on experimental evolution experiments with the marine model diatom Thalassisoria pseudonana that thermal tolerance can rapidly evolve within 300 generations. Our results indicate that upper and lower temperature limits were fixed, however temperature optima for growth shifted towards the selection temperature. Furthermore, temperature had a significant impact on average cell diameter, bSi content and cellular stoichiometry (C:N:P). Physiological adaptation to high temperature was underpinned by differential expression of genes related to protein metabolism (protein binding and folding), and down-regulation of mismatch repair mechanisms potentially causing a high number of SNPs in the genome. Furthermore, several transposable elements showed strong, temperature specific up-regulation suggesting epigenetic enabled genome plasticity. Our results highlight the relation of adaptive pheno- and genotypes driven by temperature selection. This knowledge is key to our understanding of how the environment shapes the evolution of microbes and the biogeochemical processes they drive.