Following surveys of seaweeds in Oregon and the Caribbean, four algal species were selected for study on the basis of their crude extract biological activity or thin-layer chromatographic characteristics. Extracts were fractionated by normal phase chromatography, including high performance liquid chromatography (HPLC). Derivatives (methyl esters, acetates, and benzoates) of natural products were produced to facilitate isolation or structure elucidation efforts. Structure proofs utilized high-field nuclear magnetic resonance (NMR) spectroscopy, including hetero- and homo-nuclear two-dimensional experiments. Infrared and ultraviolet spectra, optical rotations, mass spectra, and circular dichroic spectroscopy were also employed. The Puerto Rican red alga Murrayella periclados was found to contain (12S)-hydroxyeicosapentaenoic and (12S)-hydroxyeicosatetraenoic acids, two mammalian autacoids for which new biological activities are reported. Further eicosanoid metabolism was demonstrated by the isolation of (6E)-leukotriene B₄ and two diastereomers each of the insulin-release mediators hepoxilin B₃ and B₄. The Oregon green alga Acrosiphonia coalita also was found to contain novel fatty acid-derived substances. Some of these contain an unprecedented structural feature, a conjugated trienal in which the aldehyde moiety branches off from the fatty acid chain. Exciton chirality studies of benzoate derivatives indicate that the alga introduces oxygenation in the 9R, 13S, and 16S positions on precursor polyunsaturated fatty acid chains. The Oregon red alga Laurencia spectabilis was found to produce an equilibrium mixture of (±)-2-hydroxy-2-methyldihydrofuran-3-one and 5-hydroxy- 2,3-pentanedione, which dimerizes on silica gel to produce spiro-bis-pinnaketal, a compound which was previously reported from L. pinnatifida and is probably an artifact of isolation. The Oregon red alga Prionitis lanceolata displays high levels of apparent tryptophan metabolism. Never before isolated from a plant or marine source, 3- (hydroxyacetyl)indole, active at 10⁻¹⁰ M in the lettuce-seedling root elongation assay, was isolated in 0.07% yield along with indole-3-carboxaldehyde (0.5%) and indole-3-carboxylate (0.04% ). / Graduation date: 1992

No description available.

I have participated in a drug discovery program designed to screen marine algae for inhibitors of cancer-related enzymes, antitumor compounds, antiinflammatory substances, and other agents of potential pharmaceutical utility. Over 1,500 lipid and aqueous extracts of marine plants and animals were surveyed for biomedical potential. Assays designed to screen extracts for new types of marine toxins have served to guide the isolation and identification of biologically active compounds. Extracts of the Oregon marine alga Constantinea simplex were found to contain a mixture of constanolactones, and lactonized cyclopropyl-containing oxylipin metabolites that logically derive from arachidonic and eicosapentaenoic acids. Spectroscopic analysis and chiroptical measurements of the natural products and various synthetically produced derivatives afforded the structures of seven structurally related compounds. Nakienones A-C and nakitriol, a series of reactive cytotoxic metabolites, were isolated from dead and necrotic branches of stony coral (Acropora sp.) which were completely covered with a gray-black mat of cyanobacteria (Synechocystis sp.). Their structures were determined spectroscopically by interpretation of 2D-NMR experiments, including heteronuclear multiple-bond coherence spectroscopy (HMBC) and 2-D nuclear Overhauser exchange spectroscopy (NOESY), and by comparison with model compounds. Bioassay-guided fractionation of the organic extract of a Curacao Lyngbya majuscula organic extract led to the isolation of an extremely potent brine shrimp toxin with antiproliferative activity. The structure of this new thiazoline ring-containing lipid, curacin A, was deduced from spectroscopic information and comparison of products obtained from chemical degradation of the natural product with the same substances prepared by synthesis. Curacin A is an antimitotic agent that inhibits microtubule assembly and the binding of colchicine to tubulin. In addition to curacin A, a potent new ichthyotoxic depsipeptide (antillatoxin), a new malyngamide derivative, and an unusual molluscicidal compound have been isolated from this alga. / Graduation date: 1995

Halogenated monoterpene production by the red macroalga Octhodes secundiramea was studied-under different levels of nutrient, bromide, vanadium and light delivery. The key products formed were identified as myrcene, betaelemene, the Z and E isomers of l0-bromomyrcene, 3-chloro-10E-bromomyrcene, Apakaocthodene A, and isomers of chloromyrcene and dibromomyrcene. The proposed pathway begins with an initial halogenation of myrcene to 3- chloromyrcene or 10-bromomyrcene. 10-bromomyrcene reacts with either chloride or bromide ions producing either 3-chloro-10E-bromomyrcene or 1,10- dibromomyrcene. The production of Apakaochtodene A starts from myrcene with 4 consecutive halogenation reactions, the first one forming the cyclic ring. All fermentations were carried out in similar 2L photobioreactors. Temperature was controlled at 26°C, with a photoperiod of 14 hours on:10 hours off. Incident light intensity was 143 μE m⁻² s⁻¹ for nutrient, bromide and vanadium delivery cultivations and the mean light intensity was set to 52.5, 125, and 240 μE m⁻² s⁻¹ for light delivery cultivations. The pH was buffered to 8.0 with sodium bicarbonate, but not controlled. Air was supplied at 630 mL min⁻¹, vvm of 0.3, and CO₂ was supplied at 2 mL min⁻¹ for nutrient and light delivery cultivations and 0 mL min⁻¹ for bromide and vanadium delivery cultivations. For nutrient delivery cultivations, nitrate was delivered at 0.006, 0.076 and 0.74 mmol Nitrate L⁻¹ day⁻¹ and phosphate was delivered at 0.33, 3.99 and 38.4 μmol Phosphate L⁻¹ day⁻¹. For semi-continuous delivery cultivations, bromide was delivered at 0.0, 0.0074, 0.013 and 0.13 mmol Bromide L⁻¹ day⁻¹ at a constant vanadium delivery of 6.38 to 7.35 nmol Vanadium L⁻¹ day⁻¹, and vanadium delivery was set to 0.0 and 6.38 nmol Vanadium L⁻¹ day⁻' at a constant bromide delivery of 0.13 mmol Bromide L⁻¹ day⁻¹. For fed-batch bromide and vanadium delivery cultivations the one time pulse of bromide raised the medium bromide concentration to 0.0, 0.038, 0.128 and 1.4 mmol Bromide L⁻¹, with the vanadium concentration going to 416 to 520 nmol Vandadium L⁻¹, and the pulse of vanadium raised the medium vanadium concentration to 0.0 and 515 mmol Vanadium L⁻¹, with the bromide concentration going to 1.1 to 1.4 mmol Bromide L⁻¹. / Graduation date: 2005

Graduation date: 1998

This thesis is an account of investigation on the natural products deriving from various marine algae and has resulted in the discovery of eleven novel bioactive metabolites. Isolation and characterization of these new molecules were carried out using different chromatographic techniques and by analyses of different spectroscopic data, respectively. Using bioassay guided fractionation (brine shrimp toxicity assay), I isolated and identified five new, biologically active compounds [2β,3α-epitaondiol, flabellinol, flabellinone, stypoaldehyde and stypohydroperoxide], together with five known compounds [2-geranylgeranyl-6-methyl-1, 4-benzoquinone, (-) epistypodiol, (-) stypoldione, fucoxanthin and iditol] from the marine brown alga Stypopodium flabelliforme, collected from Papua New Guinea. All of the new compounds were found to have cytotoxic activity (EC₅₀ ranges from 0.8-10 μg/ml) in human lung cancer (NCI-H460). 2β,3α-epitaondiol and flabellinol exhibited strong sodium channel blocking activity (EC₅₀=0.3 and 0.9 μg/ml, respectively). As a result of efforts to identify bioactive agents from marine algae, I have isolated and identified one new halogenated monoterpene [(-)-(5E,7Z)-3,4,8- trichloro-7-dichloromethyl-3-methyl-1,5,7-octatriene] in addition to another three known halogenated monoterpene compounds from the red alga Plocamium cartilagineum collected from the eastern coast of South Africa. [(-)-(5E,7Z)-3,4,8- trichloro-7-dichloromethyl-3-methyl-1,5,7-octatriene] was found to be active as a cytotoxic agent in human lung cancer (NCI-H460) and mouse neuro-2a cell lines (EC₅₀ 4 μg/ml). As part of continued search for bioactive secondary metabolites from marine sources using a bioassay guided fractionation approach (anti-trypanosome activity), I examined the organic extract of a Papua New Guinean collection of the green alga Udotea orientalis growing on a coral wall and collected in September 1998. Successive HPLC separations resulted in the isolation of three new compounds; (+) curcuepoxide A, (+) curcuepoxide B and (+)-l0α-hydroxycurcudiol. In addition I isolated four known compounds; (+)-10β- hydroxycurcudiol, (+) curcuphenol, (+) curcudiol and (+) curcudiol-10-one. A bioassay guided investigation approach (anti-Sirt2) of a Lyngbya majuscula collection from Key West Florida, led to the discovery of two novel bioactive natural products [(+)-malyngamide X and one cyclic depsipeptide, (+)-floridamide]. The new cyclic depsipeptide, (+)-floridamide contains four amino acids units beside the unique unit, 2,2-dimethyl-3-hydroxyoctanoic acid (Dhoaa). / Graduation date: 2004

Thesis (Ph. D.)--Oregon State University, 2006. / Printout. Includes bibliographical references (leaves 223-233). Also available on the World Wide Web.

Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Durban University of Technology, Durban, South Africa, 2015. / Fossil fuel reserves have been diminishing worldwide thus making them very scarce in the long term. These fuel sources and their by-products which are used commercially tend to produce large quantities of emissions. Some of them are believed to be toxic to flora and fauna. It is primarily for this reason that researchers worldwide have begun to seek out alternative sources of environmentally safe fuel. Biodiesel from algae is one of these sources that have been examined over the last few decades. Biodiesel has been produced from other plant-based material and waste oils in countries like America and Japan. However, the use of food based crops for biodiesel production has been challenged as it has an impact on food production on an international scale. Algae have only recently been investigated for their feasibility for biodiesel production on a large scale. The aim of this study was to investigate and develop technologies for biodiesel production from algae. The species of algae chosen were chlorella sp and scenedesmus sp., since they are indigeneous to Kwazulu Natal in South Africa. Samples were obtained from a local raceway pond and prepared for analysis. Drying protocols used freeze, oven and sun drying for initial preparation of the samples for analysis. Sun drying was the least energy intensive but most time consuming. At laboratory scale, oven drying was chosen as the best alternative. Lipid extraction methods investigated were the separating funnel method, the soxhlet method, microwave assisted extraction (MAE) and the expeller press. Thirteen solvents covering a range of polarities were used with the extraction methods to determine the efficiency of the solvent with these methods. Optimization of the MAE method was conducted using both the one factor at a time (OFAT) method and a design of experiment (DOE) statistical method. The shelf life of algal biomass was determined by ageing the samples for approximately three months. Direct and in-situ transesterification of lipid extracts to produce biodiesel was investigated using both acid and base catalysis. Qualitative and quantitative analyses were conducted using Fourier transform infra-red (FTIR) and gas chromatography (GC). Chemical and physical characterization of the biodiesel produced from the algal lipid extracts were compared to both local and international standard specifications for biodiesel. In terms of extraction efficiency, it was found that soxhlet and microwave assisted extraction methods were almost equally good. This was proved by the MAE method yielding an average of 10.0% lipids for chloroform, ethanol and hexane after 30 mL of solvent was used in an extraction time of 10 minutes, while the soxhlet method yielded 10.36% lipids using an extraction volume of 100 mL of solvent with an extraction time of 3 hours. Chloroform, ethanol and hexane were more efficient than the other ten solvents used. This was shown by these three solvents producing lipid quantities between 10% to 11% while all the other solvents produced lipid quantities between 2 and 10 %. The best extraction efficiency was achieved by the binary solvent mixture made up of chloroform and ethanol in a 1:1 ratio. Under the conditions optimized, this solvent ratio yielded a lipid content of 11.76%. The methods chosen and optimized for extraction are very efficient, but the actual cost of production of biodiesel need to be determined. Physical methods like the expeller press are not feasible for extraction of the type of biomass produced unless algae are pelletized to improve extraction. This will impact on the cost of producing biodiesel. The transesterification protocols investigated show that the base catalysis produced biodiesel with a ratio of saturates to unsaturates conducive to a good fuel product. The direct esterification method in this study proved to be better than the in-situ method for biodiesel production. The in-situ method was also more labour intensive. Chromatography was found to be a fast and efficient method for qualitative and quantitative determination of biodiesel. Characterization tests showed that the quality of biodiesel produced was satisfactory. It also showed that the methods used in this study were feasible for the satisfactory production of biodiesel which meets local and international specifications.

Microalgae have a variety of commercial applications, the oldest of which include utilisation as a food source and for use in wastewater treatment. These applications, however, are seldom combined due to toxicity concerns, for ethical reasons, and generally the requirement for cultivation of a single algae species for use as a feed supplement. These problems might be negated if a “safer” wastewater such as that from agricultural and/or commercial food production facilities were to be utilised and if a stable algae population can be maintained. In this investigation preliminary studies were carried out using an Integrated Algae Pond System (IAPS) for domestic wastewater treatment to determine the species composition in the associated High Rate Algae Ponds (HRAPs). The effect of different modes of operation, continuous versus batch, on nutrient removal, productivity and species composition was also investigated. Furthermore, indigenous species in the HRAP were isolated and molecularly identified as, Chlorella, Micractinium, Scenedesmus and Pediastrum. Additionally, the effect of the nor amino acid, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA) and its Cu-chelated derivative, on the growth and biochemical composition of Chlorella, Micractinium, Scenedesmus, Pediastrum and Spirulina was investigated. Species composition in the HRAP was stable under continuous operation with Micractinium dominating > 90% of the algae population. Under batch operation the population dynamic shifted; Chlorella outcompeted Micractinium possibly due to nutrient depletion and selective grazing pressures caused by proliferation of Daphnia. Higher species diversity was observed during batch mode as slower growing algae were able to establish in the HRAP. Nutrient removal efficiency and biomass productivity was higher in continuous mode, however lower nutrient levels were obtained in batch operation. HMTBA did not significantly affect growth rate, however treatment with 10 mg.L-1 resulted in slightly increased growth rate in Micractinium and increased final biomass concentrations in Chlorella, Micractinium and Spirulina (although this was not statistically significant for Micractinium and Spirulina), which are known mixotrophic species. Algae treated with Cu-HMTBA, showed reduced final biomass concentration with 10 mg.L-1, caused by Cu toxicity. Biochemical composition of the algae was species-specific and differed through the growth cycle, with high protein observed during early growth and high carbohydrate during late growth/early stationary phase. Additionally, 0.1 mg.L-1 HMTBA and Cu-HMTBA significantly reduced protein content in Chlorella, Micractinium, Scenedesmus and Pediastrum. In conclusion, operation of the HRAP in continuous culture provided suitable wastewater treatment with high productivity of an ideal species, Micractinium, for use in animal feed supplementation. This species had 40% protein content during growth (higher than the other species tested) and dominated the HRAP at > 90% of the algae population during continuous mode. Addition of HMTBA (> 1 mg.L-1) to algae cultivation systems and those treating wastewater, has the potential to improve productivity and the value of the biomass by enhancing protein content. Overall, the co-utilisation of microalgae for wastewater treatment and the generation of a biomass rich in protein, for incorporation into formulated animal feed supplements, represents a closed ecosystem which conserves nutrients and regenerates a most valuable resource, water.

This thesis is an investigation of the natural products deriving from marine algae and cyanobacteria and has resulted in the discovery of eleven new secondary metabolites. The structure elucidations of these new molecules were performed using a variety of spectroscopic techniques. Four new macrolides were isolated and characterized from the Madagascar marine cyanobacterium Geitlerinema sp. These ankaraholides are structurally similar to the potently cytotoxic swinholides and were found to have cytotoxicities ranging from 178 nM to 354 nM against human lung cancer (NCI-H460) and mouse neuro-2a cell lines. Since swinholide-type compounds were previously localized to the heterotrophic bacteria of sponges, these findings raise intriguing questions about their true metabolic source. Geitlerinema sp. was found to be particularly rich in chemistry, and also produced the new linear lipopeptide mitsoamide with unusual structural features including an aminal moiety, a homolysine residue and a polyketide unit (3,7- dimethoxy-5-methyl- nonanedioic acid) (DMNA). A collection of the red marine alga Portieria hornemannii from the south of Madagascar (Tolagniaro, Fort Dauphin), led to the isolation of the previously reported halogenated monoterpene, halomon, and the discovery of three new related metabolites. These molecules were found to inhibit DNA methyltransferase 1 (DNMT-1). As a result of efforts to identify bioactive agents from the marine cyanobacterium Lyngbya majuscula, tanikolide dimer, a novel SIRT2 inhibitor (IC50 = 176 nM), and tanikolide seco-acid were isolated. The depside molecular structure of tanikolide dimer, which is likely a meso compound, was established by NMR, MS and chiral HPLC analyses. The structure of tanikolide dimer raises a number of intriguing configurational and biosynthetic questions for further study. The bioassay guided fractionation of a collection of the brown marine alga Dictyota sp. from Netherland Antilles Playa Fort, led to the identification of a novel HDAC inhibitor with a dolastane carbon skeleton. The novel molecule was also found to possess antimalarial activity. Other known HDAC inhibitors with interesting antimalarial activity have been reported previously, and based on this efficacy against malaria, HDAC appears to be a viable target for the development of antiparasitic agents. / Graduation date: 2006