Thesis (M.S.)--University of North Carolina Wilmington, 2007. / Includes bibliographical references (leaves: 43-46)

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 71-79). Also available via the World Wide Web.

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Driven by the increase in industrialization and population, the global demand of energy and material products is steadily growing. Microalgae have come into prominence in the past several decades due to their ability to utilize solar energy to fix atmospheric carbon dioxide, and produce biomass and lipids at productivities much higher than those possible with terrestrial biomass. The main objective of this research is to maximize the biomass and lipid production of Chlorella vulgaris by varying different external conditions so as to achieve the ideal feedstock for the production of commodity chemicals and implement wastewater treatment. The effects of various culture medium compositions on Chlorella vulgaris growth and lipid production were investigated using batch culture. Thirteen culture media: Modified Chu’s No. 10, Bold basal, BG-11, Modified BG-11, N-8, M-8, RM, Modified Spirulina, F-si, Fogg’s Nitrogen free, Fog, F/2, and Johnson medium were compared in terms of optical density, biomass production, specific growth rate and lipid production. Following a 10-day culture in a temperature controlled environment, Bold basal medium was found to have the highest average biomass productivity of 48.056 ± 2.097 mg L -1 day -1 , with overall specific growth rate of (d -1 ): 0.211 ± 0.003 and lipid productivity of 9.295 mg L -1 day -1 among the selected media. This is a basis for the optimization of different cultivating medium to enhance algal lipid production. In order to maximize the quality and quantity of the algal biomass and lipid content in Chlorella vulgaris, different strategies were used using different ratios of nitrogen and phosphorus source in the modified Bold basal medium (BBM). In the 12-day batch culture period, the highest biomass productivity obtained was 72.083 mg L -1 day -1 under Bold basal medium with Nitrogem control Phosphorus limited conditions. The highest lipid content, lipid concentration and lipid productivity obtained were 53.202%, 287.291 mg/L and 23.449 mg L -1 day -1 respectively, under Bold basal medium with Nitrogen Control Phosphorus Deprivation conditions. Nitrogen starvation was found to be the critical factor affecting the biomass production and lipid accumulation while the starvation of phosphorus induced a higher total lipid content and affected the lipid composition of Chlorella vulgaris cultures. Recently, as the demand for pure microalgae strains for the production of algal lipid as a feedstock of renewable energy has been increasing, the designation of an effective photobioreactor (PBR) for mass cultivation is essential to assure stability in the amount of feedstock. Various PBRs design such as bubbling, air-lift, porous air-lift was compared. In general, the bubbling design is a better PBR designs than the others, having the highest biomass concentration of 0.78 g/L during the culture time. Besides, it was observed that the 35 cm draft tube of the porous air-lift PBR had shorter mixing time (24.5 seconds) and higher biomass concentration (0.518 g/L) than the 50 cm air- lift design. The bubbling PBR with the highest gas flowrate of 2.7 L/min produced the highest biomass production of 0.74 g/L within the cultivation time. The information is shown to be a useful guide for determining the optimal condition of the PBRs. Light wavelengths and intensities were determinant factors in affecting the growth and lipid content of autotrophic organisms such as C. vulgaris. The experiment investigated the effect of algal lipid production by using LEDs (Light Emitting Diodes) with different wavelengths. C. vulgaris was grown in the effluent for 10 days under the photoperiod of 18:6 h Light/Dark cycles with different visible light sources (cool white, blue and red) and intensities (50 μmol m -2 s -1 ) at 25°C. The overall maximum dry biomass of 1353.33 mg/L was observed at 50 μmol m -2 s -1 cool white light during 10th culture day, with the highest overall productivity of dry biomass production (117.23 mg/L d -1 ) within cultivation time. The highest lipid content (34.06 %) was obtained with the blue color due to light efficiency and deep penetration to the photosynthetic pigments (chlorophyll) in C. vulgaris. However, the highest lipid productivity was observed in cool white light of 318.63 mg/L during the 10th culture day. The effect of light intensity toward the lipid productivity was further investigated by increasing the light intensity of cool white light. The highest lipid productivity was observed at 110 μmol m -2 s -1 in a light intensity of 658.99 mg/L during the 10th culture day. In high irradiance (110 μmol m -2 s -1 ), the proportion of poly unsaturated fatty acid (C18:1 and C18:2) contributed most of the fatty acid methyl ester (FAME) content in the collected sample, irrespective of all treatments. The next study optimized the harvesting rate of algae by using an electro- coagulation-flotation (ECF) harvester, which combines the electrochemical reaction in the electrodes and the dispersion of hydrogen gas to allow floatation of microalgae cell for surface harvesting. The response surface methodology model (RSM) was employed to optimize different ECF parameters: electrode plate material, electrode plate number, charge of electrode, electrolyte concentration and pH of the solution. The model revealed that aluminum was the best electrode material for the ECF process. It was also found that a three electrode plates setup with one anode and two cathodes had the best performance for harvesting. Additionally, sodium chloride (NaCl) at 8 g/L in harvesting medium could increase the flocculant concentration and reduce electric power consumption. Also, having the culture medium at pH 4 also had a significant effect on improving the flocculant production. Combining these optimal conditions, the highest flocculant concentration reached 2966 mg/L in 60 mins; a 79.8% increase in flocculant concentration, based on the tested conditions. The results of this study show the significance of different parameters affecting the coagulation and flocculation of C. vulgaris and provide a reference for the design of a large-scaled harvester for microalgae harvesting in the further study. To conclude, this research comprises a study on the use of indigenous algae for the production of algal lipid, which is used to produce commodity chemicals. Details on the use of nutrient sources, the techniques of cultivation and the optimization of cell harvesting were included so as to remove nutrients from effluents to minimize the occurrence of eutrophication in harbor, thereby providing economic advantages. Thus, the optimization of these processes is very adequate and offers significant advantages for the wastewater treatment. The developing of algal cell biotechnology is necessary to further enhance algal lipid production in an attempt to apply it commercially.

Morphogenesis in walled organisms represents a highly controlled process by which the variability of shapes arises through changes in the structure and mechanics of the cell wall. Despite taking different evolutionary paths, land plants and some brown algae exhibit great developmental and morphological similarities. In two brown algal model systems: the Sargassum muticum apex and the Fucus serratus embryo, I have used a combination of imaging techniques, growth analyses, surgical and pharmacological treatments, as well as molecular, biochemical and mechanical approaches to characterise the growth patterns and the cell wall contribution to shape change. To understand how the adult algal body is formed, I examined the branching strategy (phyllotaxis) in S. muticum. My results suggest that in S. muticum the spiral phyllotactic pattern and the apical cell division pattern are not linked. The phytohormone auxin and the biochemical changes of the cell wall do not seem to be correlated with the bud outgrowth, contrary to observations in plants. In summary, these results suggest Sargassum convergently developed a distinct growth mechanism with similar shape outcome as observed in plants. This dissertation is one of the first attempts to explore cell wall mechanics in brown algal development and its correlation with underlying cell wall biochemistry utilising the Fucus embryo as a known system. The results suggest a correlation between the wall mechanics and alginate biochemistry with the growing and non-growing regions of the embryo. In addition, altering cell wall deposition or composition has a strong effect on embryo rhizoid elongation and is, in certain cases, accompanied by significant increase in cell wall stiffness and reduction of alginate epitopes. Furthermore, preliminary results exploring transcriptomic changes during development indicate differential expression of particular alginate biosynthesis enzymes (mannuronan C5 epimerases) during development, suggesting alginate conformational modifications might be stage specific. These results contribute to the current knowledge addressing the importance of cell walls in brown algal development using novel tools and approaches. Understanding developmental processes in brown algae will provide a better insight how similar morphogenetic traits are established using different body-building mechanisms.

Microalgae have a profound effect on the world due to their large contribution to net carbon fixation. Although they are phototrophic, more than 50% of microalgae are thought to depend on external supply of metabolites such as B-vitamins. In oceans, algae are therefore often found together with a community of bacteria and form intricate networks where metabolites are exchanged. Currently, only a fraction of the related mechanisms and metabolite exchanges between algae and bacteria have been uncovered and many more are likely to exist. The work presented in this thesis is based on a model system for algae-bacterial interactions made up of the green alga, Lobomonas rostrata and the alpha-proteobacterium Mesorhizobium loti. In the model system, it is known that the bacterium provides vitamin B12 to the alga and itself, whilst the alga provides fixed carbon. I have applied methods from the field of computational biology to study the interactions between these organisms and other similar partnerships, with the aim of uncovering new insights. The thesis is made up of three research chapters, each focused on using a specific method to study algae-bacterial interactions. I developed a genome scale metabolic model of metabolism of M. loti that enabled simulation of growth. The model simulates 1908 enzymatic reactions and takes 1804 metabolites into account. Using the model, I simulated growth of the bacterium on 1018 different substrates with the aim of identifying substrates supplied by L. rostrata when the two organisms are co-cultured. In addition, I carried out a set of simulations studying the bacterium’s ability to produce B12 from 1368 different substrates. The modelling efforts in this project was successful in enabling simulations, but it was not possible to validate the simulations with experimental data. A transcriptomics experiment was undertaken with the aim of identifying genes related to the interaction between L. rostrata and M. loti. In the experiment, the partners from the model system was grown in axenic and co-culture conditions and RNA samples were taken from each state. Using RNA-seq, the RNA samples were sequenced and from this a candidate transcriptome was created. The expression of each putative gene was then quantified and differentially expressed genes were identified. Based on sequence similarity, candidate functions were assigned where possible. In the analysis of differentially expressed genes, it was found that there appears to be an increased expression of a transporter responsible for uptake of the plant hormone, auxin. Currently, only a small fraction of all bacteria has been shown to produce B12 and it is not clear in which phylogenetic groups this is a common trait. I therefore applied methods from comparative genomics to study the synthesis of this metabolite in more than 8000 bacterial species. This involved developing a computational framework that allowed me to search for the presence of more than 50 genes in more than 8000 genomes in a rapid manner. I found that 37.2% of bacteria can synthesis B12 and that this capability is very common in some phylogenetic groups such as Cyanobacteria, but extremely rare in others such as Lactobacillus. I was also able to confirm that cyanobacteria are not able to make cobalamin, a variant of B12 used by eukaryotic algae, and thus they are unlikely to support algal growth in the photic zone. In the final section of the thesis, I discuss the application of computational biology methods in this field and summarise my experience from applying genome scale modelling, comparative genomics and transcriptomics to study algae-bacterial interactions.

Chromophoric Dissolved Organic Matter (CDOM) is a substance produced by the planktonic community that naturally blocks biologically damaging, ultraviolet radiation (UV; wavelengths = 280 to 400 nm). While a variety of planktonic species produce CDOM, investigations into the quality and quantity of their CDOM production are few. The purpose of this study was to investigate the properties of CDOM produced by phytoplankton versus zooplankton. Typically, UV radiation breaks down CDOM. However, marine organisms sometimes produce CDOM that, when exposed to sunlight, increases its absorbance of UV radiation. The reasons for this anomaly and the source of this unusual CDOM are unclear. To test for distinctions among sources, filtered water samples from cultures of two marine phytoplankton, Gymnodinium sp. and Dunaliella sp., and a marine copepod, Tigriopus californicus were exposed to sunlight for intervals of 0, 0.5, 1, 2, and 4 hours. The maximal UVB radiation (wavelengths; λ = 280 to 320 nm) was 0.1699 J cm-2. Before and after sunlight exposures, CDOM sources were assessed by comparing absorbance spectra from 184 to 730 nm with detailed comparisons at wavelengths of 254, 305, 320, and 350 nm. Results are expressed as "absorption", which is the raw absorbance converted to the natural log. Before exposure to sunlight, CDOM from algal species had 5- to 8-times higher average absorption values across UV wavelengths than CDOM produced by Tigriopus. CDOM samples produced by phytoplankton were more susceptible to photochemical change than CDOM from Tigriopus. In response to sunlight, CDOM from Gymnodinium varied greatly, increasing in UV absorption in 2 out of 5 replicates but decreasing in absorption in the other replicates. In contrast, peak absorption at λ = 260 nm of CDOM from Dunaliella decreased directionally by an average of 76% in absorption and 75% in specific absorption, and this decrease in absorption was significant (p = 0.03). CDOM from Tigriopus significantly decreased in both absorption (all UV wavelengths, p < 0.01) and "specific absorption", which is absorption standardized per mg of dissolved organic carbon (λ = 320 nm; p < 0.03), but the magnitude of change was only 17%. Thus, photochemical responses of CDOM from the three planktonic sources demonstrated more, consistently less, and unchanged UV absorption. Phytoplankton biomass, which is greater than that of copepods at the next trophic level, likely produce more CDOM but because of greater variability in CDOM absorbance of UV both within and between algal sources, copepods may offer a more stable source of UV protection depending on sunlight exposure and their relative abundance. Definitive conclusions depend on future laboratory studies expanded to more planktonic organisms as well as in situ studies during oceanographic cruises to test the relative contributions of planktonic species.

Bacterial concentrations in natural sea water were significantly reduced by 5ptm filtration and subsequent treatment with ozone and UV light led to further reductions in total viable counts of bacteria and autoclaving water gave complete sterility. However, with the addition of artificial diets, such treated water was recolonised very rapidly and within 24h, these water samples had significantly higher bacteria counts than the 5pm filtered sea water treatment (f = 21.30; p<0.001). Further treatment of 5ýtrn filtered sea water also led to changes in the bacterial composition of the water. In ozonated and UV light irradiated water samples, the proportions of presumptive Vibrid sp. increased in the 24h following treatment while in 5ýLm filtered and natural sea water samples, the proportions of presumptive Vibrid sp. fell over the same period. When Penaeus indicus PZ1 stage larvae were reared to PI-1 stage in 5lam filtered sea water, good larval survival was obtained irrespective of whether live or microencapsulated artificial diets were fed. In contrast, larvae raised in autoclaved, ozonated and UV light irradiated water exhibited significantly lower survival when raised on live diets and often showed complete mortalities when fed on microencapsulated diets. In periods of poor sea water quality, additions of bacteria from algal cultures to PZ1 stage larvae fed algae had no effect on larval survival to M1 stage for larvae reared in 5gm filtered sea water or autoclaved water. In contrast, additions of these bacteria to larvae reared in 5pm filtered sea water and fed on MED, led to a significant increase in larval survival. This effect was absent when larvae were reared in autoclaved water. Addition of bacteria from algal cultures were found to inhibit counts of presumed viable Vibrid sp. while having no effects on total viable counts. No significant differences were observed in the percentage of soluble protein leached from microencapsulated diets and micro particulate diets when incubated up to 48h in bacterial laden and sterile water indicating that leaching is independent of microbial activity up to 48h. However broken microcapsules gave higher total viable bacterial counts over 48h in 5ýtm filtered sea water when compared with intact microcapsules. Significant levels of protection were conferred on larvae when either fresh or freeze-dried vaccines were administered by Immersion, but not when such vaccines were administered orally. The degree of protection offered was correlated with the virulence of the pathogen from which the vaccine was made. Enhanced protection given by vaccines produced from the more virulent strains was not wholly due to activation of the prophenoloxidase system since such vaccines induced less stimulation of the prophenoloxidase system than less virulent strains. Untreated plasma of Penaeus vannamei significantly enhanced Escherichia coli growth compared with sea water nutrient medium. In contrast, plasma from vaccinated prawns exhibited antibacterial activity detectable up to 7d after vaccination. Exposure to a mixture of fungicides Implicated in the initiation of the taura syndrome disease (TS), had no effects on the immune competence of P. indicus juveniles following live, in vivo pathogenic challenge. In addition, no effects on growth, growth rates, moulting rates or survival of postlarval and early juvenile prawns were observed following exposure to the fungicides. Prawns exhibited no gross or histopathological symptoms characteristic of TS.

Few previous studies of zooxanthellae have considered temperate Anthozoan symbioses. The present study investigates how the characteristics of zooxanthellae symbiotic with temperate Anthozoa vary in response to natural variations in environmental parameters. Variations in the number (density), division rate, size and ultrastructure of zooxanthellae from the temperate anemones Anemonia viridis (Forskal) and Anthopleura ballii (Cocks) were examined in response to season, water depth and artificial irradiance (A. viridis in aquaria). In addition, variations in chlorophyll concentrations were considered in intertidal and laboratorymaintained A. viridis. Zooxanthellae from both intertidal and shallow subtidal A. viridis showed variations which correlated with seasonal variations in environmental parameters. Zooxanthella density in intertidal A. viridis showed an inverse relationship with temperature, daylength and sunshine. Higher zooxanthella density was observed in A. viridis from a shallow, subtidal habitat during February 1998 (2.06 ± 0.11 x 108 cells g"' wet weight) than during July 1998 (1.01 ± 0.09 x 108 cells g'' wet weight; T= 7.67, p< 0.001). Stereological analysis of transmission electron micrographs showed that zooxanthellae in intertidal A. viridis had significantly higher chloroplast volume fraction during February (32.1 ± 1.5 %) than July (21.8 ± 2.1 %; T= 4.07, p<0.05). The proportion of chlorophyll a per zooxanthella was significantly higher in December than all other months except January (ANOVA, F= 5.62 p<0.05). The zooxanthellae of A. viridis may thus photoadapt to low winter irradiances by increasing zooxanthellae density, chloroplast volume and the proportion of chlorophyll a per cell. By contrast, zooxanthellae from A. viridis maintained in artificial irradiances in the laboratory of 4 µmol m=2 s' and 20 pmol m2 s' showed no variation in density or ultrastructure, due either to the low irradiances used or a lack of variation in other physical parameters compared to the field. A. ballii zooxanthella density responded to both depth and season and was lower at 6m during summer than at 6m during winter and at 18 m during both summer and winter. Chloroplast volume fractions in A. ballii was not affected by depth during winter, nor by season at 18 in. Starch and lipid stores in zooxanthellae from both A. viridis and A. ballii responded to seasonal fluctuations. Lipid was present in zooxanthellae during summer (intertidal A. viridis, volume fraction 19.8 ± 3.4 %) and absent during winter, and starch volume was significantly higher from zooxanthellae in A. ballii at 6 in in winter (14.3 ± 4.2 %) than 18 min winter (4.7 ± 1.6 %) or summer (4.7 ± 1.1 %; ANOVA, F= 6.04 p< 0.05). It is concluded that the zooxanthellae of the temperate anemones A. viridis and A. ballfi show variations in zooxanthellae characteristics which correspond to variations in dayto-day weather, season and water depth.

Seaweed has traditionally been used in Scotland and other countries both as food and for medicinal purposes, which has led to seaweed being investigated for their natural product content. Despite over 30 years of research, the majority of species found in Scotland have yet to have their chemistry examined. Extracts of seaweed were tested for antimicrobial activity against marine bacteria. Extracts of Palmaria palmata, Ulva linza, Chondrus crispus and Pelvetia canaliculata showed no detectable activity, while ethyl acetate extracts of Fucus serratus, Halidrys siliquosa, Osmundea pinnatifida and Polysiphonia fucoides showed activity against at least six of the seven strains. Extracts were screened for radical scavenging activity against ABTS, DPPH and superoxide radicals. At least one extract from each brown seaweed showed radical scavenging of at least 80 % towards ABTS+ with an ethyl acetate extract of P. fucoides and H siliquosa quenching DPPH by at least 90%. Radical scavenging activity appears to be dependent total phenolic content of extract. Extracts were subjected to a series of assays relevant to human health. Ethyl acetate extracts showed high antiparasitic activity against Trypanosoma brucei with a P. fucoides extract showing antibacterial activity toward Staphylococcus aureus. Extracts of H siliquosa and F. serratus showed cytotoxicity to Hela cells with extracts of H siliquosa showing cytotoxicity to LN CAP AS and PC 3 cell lines. An extract of H siliquosa underwent chromatography and by applying assay guided fractionation, several active fractions were identified. These were analysed using NMR and LC/MS and four compounds identified: (2E. 6E. 14E)-1-(l'-hydroxy-4'-methoxy-6'-methyl- phenyl)-5, 13-dihydroxy-12-one-3, 7, 11, 15-tetramethylhexadeca-2, 6, 14-triene, a known antibacterial compound, previously identified in H siliquosa; (2E, 6E, 10E, l4E)-1-(1'- hydroxy-4'-methoxy-6'-methyl phenyl)-5, 12 dihydroxy-3.7, 11, 15-tetramethyl hexadeca- 2.6, 10, l4-tetraene, previously identified in Cystoseira elegans, and now in H siliquosa; and two compounds that have not been reported before.