Microalgae are unicellular organisms that can be grown photoautotrophically and their abundance in natural valuable compounds makes their industrial cultivation attractive. Current technology only allows for cost-effective production of high-value compounds. Therefore, this thesis proposes the use of ecological theory and practice to improve the large-scale cultivation of low- to medium-value compounds in microalgae. In the first study a multivariate modelling approach determined the individual importance of several abiotic factors on the dynamics of a microcosm microbial community under oligotrophic and eutrophic conditions. The application of a simple model illustrated key causal relationships and demonstrated that nutrient enrichment significantly changed the relative importance of the tested abiotic variables to the dynamics of the microbial system. The second study utilised a metaproteomic approach to detail the mechanisms of co-existence and acclimation in the same microbial community. A decrease in microalgal exudation, in eutrophic conditions, affected bacterial acquisition of energy and nutrients. Furthermore, two microalgal-bacterial relationships, of potential use to synthetic ecology, were highlighted. Finally, in the third study, the competitive dynamics between two C. reinhardtii strains, a wild type and a high-lipid mutant, were studied utilising response surface methodology. In the co-culture with 25% wild type, intraspecific competition significantly increased triglyceride concentrations. The competition data also suggested there was little risk of the mutant displacing the wild type under any of the experimental treatments. Finally, the highest triglyceride productivity was found in the pure mutant culture, after just 24 hours, demonstrating potential to scale out a small batch biomanufacturing system. This thesis successfully coupled traditional ecology experiments with modern ‘omics techniques. Several existing hypotheses, regarding microalgal ecophysiology, were assessed based on their potential application in commercial microalgal cultivation. In sum, microalgal biotechnology can benefit from the integration of core principles of microalgal ecophysiology in the transition from laboratory to commercial-scale cultivation.

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The brown algae are an abundant source of renewable biomass that do not require arable land or fresh water for cultivation. Cell walls of the brown algae contain a range of unique polysaccharides with useful bioactivities; namely the sulphated fucans and alginate. The generation and characterisation of ten new monoclonal antibodies (MAbs) towards components of brown algal cell walls are described. Four MAbs, BAM1 to BAM4, recognise un-sulphated (BAM1) and sulphated (BAM2/3/4) structures of the sulphated fucans. Six MAbs, GIN1 to GIN6, recognise different structures of the alginate, ranging from high mannuronate content (GIN4) to high guluronate content (GIN6). The combination of these new MAbs, pre-existing knowledge of the brown algal cell walls and, techniques commonly used in land plant cell walls have allowed an extensive study of cell wall architecture in the brown algae. The extraction and fractionation of the cell wall has revealed that the sulphated fucans and alginate are distributed throughout the cell wall architecture and that there are differences in cell wall architecture between orders of the brown algae. Investigation into the abundance of sulphated fucans in the intertidal zone showed trends for increased sulphated fucan content higher up the intertidal zone (as determined by BAM3 and BAM4), but also revealed a ubiquitously distributed set of sulphated fucans (as determined by BAM1 and BAM2). Within tissues of the algae; sulphate fucans and alginate are spatially distributed both in terms of abundance and incorporation into the cell wall architecture – and these traits are shared between species of Fucus. Use of indirect immunohistochemistry allowed dissection of the sulphated fucan and alginate distribution in muro (in wall); revealing cell type specificity and patterning of the polysaccharides.

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This study investigated the effects of light quality, culture density, and carbon dioxide aeration on the biomass production, lipid accumulation, elemental contents, and photosynthetic pigment production of microalgae. The microalgae Chlorella vulgaris and Gloeothece membranacea were cultivated either in static (flask) or aerated and mixed (photobioreactor) cultivation modes. The highest biomass productivities in the static mode for both microalgae strains were achieved using violet luminescent filters which emitted the photosynthetic active radiation ranges. In the static mode, red wavelength ranges had a major influence on pigmentation of C.vulgaris, although they were the least efficient light condition for cultivation of G.membranacea. Red light was particularly inefficient to support growth in static cultures. Luminescent bubble column photobioreactors (PBRs) were constructed and used for the aerated mode in the blue, green, yellow, orange, and red wavelength-emitting ranges. Biomass production in both strains was enhanced in the red PBR. Lipid accumulation was significantly enhanced in high culture density groups, low light intensity, and 15% CO2 aeration conditions which all represented a form of stress to the culture. The highest biomass density of 2.12 and 2.52 g L-1 were achieved at high light intensity in the red PBR for C.vulgaris and G.membranacea respectively. The blue PBR was the least efficient light condition for biopigment production, whilst it improved lipid accumulation. Chlorophyll production in C.vulgaris was promoted by the green PBR, although it was less influenced by the light condition or culture density in G.membranacea cultures. Phycobiliproteins were the dominant pigments in G.membranacea and red light favoured synthesis of these pigments. Compared to C.vulgaris, the G.membranacea cells showed higher tolerance to 15% CO2 aeration by growing in this condition over a period of two weeks. At the 15% CO2 aeration condition G.membranacea accumulated the highest lipid content of 36.6% in the blue PBR. However, due to their lower carbon content and biomass productivity, G.membranacea was less efficient than C.vulgaris in sequestrating carbon dioxide. C.vulgaris sequestrated carbon dioxide up to 363 mg L-1 day-1 in the red PBR when aerated with 15% CO2. These results have significant implications for improved photobioreactor design for cultivation of microalgae under natural light. The proposed photobioreactor design can also tailor and improve the composition of lipids and photosynthetic compounds, using variation in light.

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Microalgae are regarded as a promising biomass resource for the production of biofuels and chemicals which does not compete with food production. Microalgae contain large amounts of lipids and have faster growth rates than terrestrial biomass. One of the current technological bottlenecks of biofuels conversion is the economic extraction and processing of microalgae components. Due to their aquatic nature microalgae contain large amounts of water when harvested. Hydrothermal liquefaction (HTL) involves processing the algae as a slurry in hot compressed water, avoiding drying of the wet feedstock. This is a major energy benefit compared to dry microalgae processing methods. A detailed characterisation of the microalgae feedstocks investigated for the current work is provided. The main differences between marine, fresh water and cyanobacteria strains are presented. The microalgae strains are investigated for biochemical composition, proximate and ultimate analysis, thermo-gravimetrical analysis, pyrolysis GC-MS, metal content, pigment analysis and by scanning electron microscopy. The results from the characterisation work are employed throughout the thesis for mass balance calculations and investigation of reaction chemistry. HTL for bio-crude production is investigated both on laboratory batch systems and a continuous pilot scale facility. Processing at mild conditions results in mainly the lipids of microalgae being extracted resulting in a high quality bio-crude. Higher temperatures are shown to result in higher yields of bio-crude as carbohydrates and proteins increasingly contribute to bio-crude formation. This allows processing of low lipid containing microalgae which are associated with higher growth rates. Maximum bio-crude yields of around 50 wt.% can be achieved but can contain significant amounts of nitrogen and oxygen. A total of 11 microalgae strains is investigated leading to an average bio-crude yield of 34 %, a heating value of 36 MJ/kg, a nitrogen content of 4.7 wt.% and an oxygen content of 13.6 wt.%. The use of homogeneous and heterogeneous catalysts is investigated to increase bio-crude quality and yields. Model compounds of protein, lipid and carbohydrates are processed individually to shed light on the HTL behaviour of microalgae components and the reaction pathways involved in bio-crude formation. The effect of sodium carbonate and formic acid as homogeneous catalysts is investigated on various microalgae strains with changing biochemical composition and on model compounds separately. It is shown that biochemical components of microalgae behave additively in respect to bio-crude formation. The trends of bio-crude formation follow lipids>protein>carbohydrates. It is further shown that carbohydrates are best processed in alkali conditions while protein and lipids are best processed without the use of catalysts. The same effect is demonstrated for algae high in carbohydrates or proteins and lipids respectively. Heterogeneous catalysts are shown not to increase the bio-crude significantly but result in additional decarboxylation of the bio-crude to reduce the oxygen level by a further 10%. The process water composition from HTL is investigated for common nutrients required for algae cultivation. It is shown that nutrients are present in higher concentrations than comparable standard algae growth media. The process water also contains large amounts of organic carbon which is considered a loss, unavailable for bio-crude formation. Growth trials in dilutions of the process water to grow fresh algae demonstrate that growth is sustainable. The organic carbon in the process water is shown to act as a substrate for mixotrophic growth resulting in increased growth rates and carbon efficiency. For analysis of the algae obtained from small scale growth trials a new analysis technique for microalgae composition analysis is introduced. This involves Py-GC-MS of model compounds and comparisons to algae pyrolysis products. Promising results are presented, showing the feasibility of detecting protein, carbohydrate and lipid levels of microalgae directly from growth cultures. Additionally the methodology is expanded to detect phytochemical concentrations such as astaxanthin and chlorophyll a. An alternative to direct hydrothermal liquefaction involving removal of valuable compounds from microalgae by hydrothermal microwave processing (HMP) is investigated. HMP is shown to remove protein and large amounts of nutrients from the algae biomass which could be used as a source of nutrients for microalgae cultivation. The cells walls are shown to be disrupted, leading to increased recovery of lipids by solvent extraction while the lipids‟ degree of saturation is not affected. This allows effective extraction of high value poly-unsaturated fatty acids. The residue form HMP is processed using flash pyrolysis and HTL for bio-crude production. The results show that bio-crudes of increased quality are produced. The technique appears especially suitable for marine microalgae strains as the salt content acts as microwave absorbers, reducing energy consumption and increasing reaction rates. Overall, the experimental work shows that hydrothermal processing is a low energy intensive wet processing technique for microalgae to produce bio-fuels and chemicals.

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