by Chow Fung-cheung, Judy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 106-117). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF ABBREVIATIONS --- p.ix / LIST OF FIGURES AND TABLES --- p.xii / Chapter SECTION I- --- ISOLATION OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1 --- Functions of Actin --- p.1 / Chapter 1.1.1 --- Functions of Actin in Animals --- p.1 / Chapter 1.1.2 --- Functions of Actin in Plants --- p.2 / Chapter 1.1.2.1 --- In Lower Plants --- p.2 / Chapter 1.1.2.2 --- In Higher Plants --- p.2 / Chapter 1.2 --- Molecular Studies of Actin Gene Families in Plants --- p.4 / Chapter 1.2.1 --- Multigene Family --- p.4 / Chapter 1.2.2 --- Homologies Across Kingdom --- p.4 / Chapter 1.2.3 --- Homologies Within Kingdom --- p.5 / Chapter 1.2.4 --- Position of Intron --- p.5 / Chapter 1.2.5 --- Differential Expression of Actin Genes --- p.7 / Chapter 1.3 --- Objectives of Present Studies --- p.7 / Chapter CHAPTER 2: --- GENERAL TECHNIQUES / Chapter 2.1 --- Growth of Algal Strain --- p.9 / Chapter 2.2 --- Growth of Bacterial Strains --- p.10 / Chapter 2.3 --- Agarose Gel Electrophoresis --- p.10 / Chapter 2.4 --- Restriction Enzyme Digestion --- p.10 / Chapter 2.5 --- Recovery of DNA Fragments from Agarose Gel --- p.11 / Chapter 2.5.1 --- Glass Powder Elution of DNA --- p.11 / Chapter 2.5.2 --- Sephaglas´ёØ BandPrep Kit --- p.11 / Chapter 2.6 --- Large Scale Preparation of Plasmid by Using Magic´ёØ Maxipreps DNA Purification System --- p.12 / Chapter 2.7 --- Ligation --- p.13 / Chapter 2.8 --- Preparation of Competent Cells --- p.13 / Chapter 2.9 --- Transformation of Competent Cells --- p.14 / Chapter 2.10 --- Screening of Recombinant Plasmids --- p.14 / Chapter 2.11 --- Spun Column Techniques --- p.15 / Chapter CHAPTER 3: --- PCR-CLONING OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter 3.1 --- Introduction --- p.16 / Chapter 3.2 --- Materials and Methods --- p.16 / Chapter 3.2.1 --- Preparation of Genomic DNA from C. vulgaris --- p.16 / Chapter 3.2.2 --- Amplification of Actin Genomic Fragments by PCR --- p.17 / Chapter 3.2.3 --- Cloning of PCR Products --- p.17 / Chapter 3.2.4 --- Southern Blotting --- p.18 / Chapter 3.2.5 --- Radiolabeling of DNA Probe --- p.19 / Chapter 3.2.6 --- Prehybridization and Hybridization --- p.19 / Chapter 3.2.7 --- Sequencing Strategies --- p.20 / Chapter 3.2.7.1 --- Isolation of Template DNA --- p.20 / Chapter 3.2.7.2 --- Template Denaturation and Primer Annealing --- p.21 / Chapter 3.2.7.3 --- Labeling and Termination Reaction --- p.21 / Chapter 3.2.7.4 --- DNA Sequencing Electrophoresis --- p.21 / Chapter 3.3 --- Results and Discussion --- p.22 / Chapter CHAPTER 4: --- CLONING OF ACTIN COMPLEMENTARY DNA FRAGMENT FROM CHLORELLA VULGARIS / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Materials and Methods --- p.31 / Chapter 4.2.1 --- Preparation of RNA --- p.31 / Chapter 4.2.2 --- RT-PCR --- p.32 / Chapter 4.2.3 --- Southern Blotting and Hybridization --- p.32 / Chapter 4.2.4 --- Radiolabeling of DNA Probe --- p.32 / Chapter 4.2.5 --- Cloning of RT-PCR Product --- p.33 / Chapter 4.2.6 --- DNA Sequencing --- p.33 / Chapter 4.2.7 --- Sequence Analysis --- p.33 / Chapter 4.3 --- Results and Discussion --- p.34 / Chapter CHAPTER 5: --- SEQUENCE COMPARISON OF ACTIN GENES / Chapter 5.1 --- Introduction --- p.44 / Chapter 5.2 --- Materials and Methods --- p.44 / Chapter 5.3 --- Results and Discussion --- p.44 / Chapter 5.3.1 --- Nucleotide Sequence Analysis --- p.44 / Chapter 5.3.2 --- Analysis of the Predicted Amino Acid Sequence --- p.49 / Chapter 5.3.3 --- Codon Usage --- p.49 / Chapter 5.3.4 --- Intron-Exon Structure in Plant Actin Genes --- p.51 / Chapter 5.3.5 --- General Discussion --- p.53 / Chapter CHAPTER 6: --- ISOLATION OF FURTHER UPSTREAM SEQUENCE FOR ACTIN GENE (CAc18G) FROM CHLORELLA VULGARIS / Chapter 6.1 --- Introduction --- p.54 / Chapter 6.2 --- Materials and Methods --- p.54 / Chapter 6.2.1 --- Genomic Southern Analysis --- p.54 / Chapter 6.2.2 --- Preparation of Actin-Enriched DNA Fraction --- p.55 / Chapter 6.2.3 --- Ligation of Actin-Enriched Fragments with Specific DNA Cassette --- p.55 / Chapter 6.2.4 --- Amplification of Upstream Sequence by Nested PCR --- p.55 / Chapter 6.2.5 --- DNA Sequencing --- p.56 / Chapter 6.3 --- Results and Discussion --- p.57 / Chapter SECTION II - --- CONSTRUCTION OF TRANSGENIC CASSETTES FOR THE PRODUCTION OF BACILLUS TOXIN IN CHLORELLA VULGARIS / Chapter CHAPTER 1： --- INTRODUCTION / Chapter 1.1 --- Characteristics of Algae --- p.64 / Chapter 1.2 --- Biotechnology Potential of Algae --- p.66 / Chapter 1.3 --- Transgenic Algae --- p.68 / Chapter 1.3.1 --- Genes of Selection for Transformant --- p.70 / Chapter 1.3.1.1 --- Homologous Genes --- p.70 / Chapter 1.3.1.2 --- Heterologous Genes --- p.70 / Chapter 1.3.2 --- Transformation Technologies Used in Algae --- p.71 / Chapter 1.3.3 --- Expression of Transgenes in Algae --- p.73 / Chapter 1.4 --- Bacillus Toxin --- p.73 / Chapter 1.4.1 --- Bacillus thuringiensis --- p.73 / Chapter 1.4.2 --- Classification of Bacillus Toxin Genes (Cry Genes) --- p.74 / Chapter 1.4.2.1 --- Type I (CtyI Genes) --- p.74 / Chapter 1.4.2.2 --- Type II (CryII Genes) --- p.75 / Chapter 1.4.2.3 --- Type III (CryIII Genes) --- p.76 / Chapter 1.4.2.4 --- Type IV (CryIV Genes) --- p.76 / Chapter 1.4.3 --- Mode of Action of Insecticidal Effects --- p.77 / Chapter 1.5 --- Insect-Resistance Transgenic Plants --- p.78 / Chapter 1.5.1 --- Transgenic Plants Expressing Crystal Protein Gene --- p.79 / Chapter 1.5.2 --- Problems Encountered --- p.80 / Chapter 1.6 --- Aims of Present Studies --- p.80 / Chapter CHAPTER 2: --- CONSTRUCTION OF TRANSGENIC CASSETTES / Chapter 2.1 --- Introduction --- p.82 / Chapter 2.2 --- Materials and Methods --- p.82 / Chapter 2.2.1 --- Preparation of Plasmids Involved in the Construction of Master Cassette --- p.82 / Chapter 2.2.2 --- Construction of Master Cassette --- p.83 / Chapter 2.2.3 --- Multiple Cloning Site (MCS) of Master Cassette --- p.83 / Chapter 2.2.4 --- Preparation of Plating Cells --- p.85 / Chapter 2.2.5 --- Titering --- p.85 / Chapter 2.2.6 --- Preparation of Plate Lysate --- p.86 / Chapter 2.2.7 --- Amplification of Coding Region of CryIVC Gene --- p.86 / Chapter 2.2.8 --- Cloning of PCR Products --- p.87 / Chapter 2.2.9 --- Construction of Transgenic Cassette --- p.87 / Chapter 2.2.10 --- Confirmation of the Junction Sites --- p.89 / Chapter 2.2.11 --- Testing for the Sensitivity of Algae Towards Kanamycin --- p.90 / Chapter 2.3 --- Results and Discussion --- p.91 / Chapter CHAPTER 3: --- TRANSFORMATION OF ALGAE BY ELECTROPORATION / Chapter 3.1 --- Introduction --- p.97 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Harvesting of Algae --- p.97 / Chapter 3.2.2 --- Electroporation at Different Field Strength --- p.98 / Chapter 3.2.3 --- Plating Culture of Algal Cells --- p.98 / Chapter 3.2.4 --- Preparation of Plasmids for Electrop oration --- p.98 / Chapter 3.2.5 --- Transformation of Algae --- p.100 / Chapter 3.2.6 --- Study on the Uptake of DNA after Electrop oration --- p.100 / Chapter 3.2.6.1 --- Genomic DNA Preparation --- p.100 / Chapter 3.2.6.2 --- Analysis of DNA Uptake --- p.101 / Chapter 3.3 --- Results and Discussion --- p.101 / REFERENCES --- p.106 / APPENDIX --- p.118

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1989. / Includes bibliographical references (leaves 159-172) / Microfiche. / xvii, 172 leaves, bound ill. 29 cm

No description available.

abstract: Recognition of algae as a “Fit for Purpose” biomass and its potential as an energy and bio-product resource remains relatively obscure. This is due to the absence of tailored and unified production information necessary to overcome several barriers for commercial viability and environmental sustainability. The purpose of this research was to provide experimentally verifiable estimates for direct energy and water demand for the algal cultivation stage which yields algal biomass for biofuels and other bio-products. Algal biomass productivity was evaluated using different cultivation methods in conjunction with assessment for potential reduction in energy and water consumption for production of fuel and feed. Direct water and energy demands are the major focal sustainability metrics in hot and arid climates and are influenced by environmental and operational variables connected with selected algal cultivation technologies. Evaporation is a key component of direct water demand for algal cultivation and directly related to variations in temperature and relative humidity. Temperature control strategies relative to design and operational variables were necessary to mitigate overheating of the outdoor algae culture in panel photobioreactors and sub-optimal cultivation temperature in open pond raceways. Mixing in cultivation systems was a major component in direct energy demand that was provided by aeration in panel bioreactors and paddlewheels in open pond raceways. Management of aeration time to meet required biological interactions provides opportunities for reduced direct energy demand in panel photobioreactors. However, the potential for reduction in direct energy demand in raceway ponds is limited to hydraulics and head loss. Algal cultivation systems were reviewed for potential integration into dairy facilities in order to determine direct energy demand and nutrient requirements for algal biomass production for animal feed. The direct energy assessment was also evaluated for key components of related energy and design parameters for conventional raceway ponds and a gravity fed system. The results of this research provide a platform for selecting appropriate production scenarios with respect to resource use and to ensure a cost effective product with the least environmental burden. / Dissertation/Thesis / Doctoral Dissertation Design 2015

The development of the High Rate Algal Pond (HRAP) as an independent tertiary treatment unit operation for phosphate and nitrate removal is reported. A novel Integrated Algal Ponding System (lAPS) design is proposed for nutrient removal from the effluents of both a conventional domestic sewage treatment plant and from an Advanced Integrated Wastewater Ponding System (AIWPS). The viability of an independently operated HRAP has been identified and termed the Independent High Rate Algal Pond (l-HRAP). A 500 m² pilot 1- HRAP was operated in such a way as to facilitate the precipitation of calcium phosphate, known to be controlled by pH (greater than 9.4) and resulting in final phosphate levels of less than 1 mg.L⁻¹ as P0₄-P. The incorporation of the I-HRAP into a denitrification process was also investigated. Continuously fed column reactors, utilising algal biomass as a carbon source, showed that the heterotrophic bacterial community dominant in the anaerobic algal sludge were denitrifying the nitrate in the feed. It was demonstrated that as the cultures were stressed (using increased nitrate concentrations, anaerobiosis and light starvation) total polysaccharide (TPS) concentrations increased, with a notable increase 111 the exopolysaccharide (EPS) fraction. These experiments corroborated the hypothesis that harvested microalgal biomass can be manipulated to produce, and release, exopolymeric substances under stress conditions, and which may serve as carbon source for denitrification. In both batch flask studies and in laboratory-scale reactor systems, harvested microalgal biomass from an HRAP was shown to produce exopolymeric substances under stress conditions. Initial high loading-rates of greater than 20 mg.L⁻¹ NO₃-N resulted in double the amount of exopolysaccharide production than in flasks with initial low loading-rates (less than 5 mg.L⁻¹ NO₃-N). Making use of an upflow anaerobic sludge blanket-type degrading-bed reactor, and an anaerobic, flooded trickle filter (ANTRIC) receiving HRAP effluent, the relationship between denitrification and the changes in polysaccharide content was investigated. This phenomenon has considerable beneficial implications in biological wastewater treatment systems where high nitrate concentration in the final effluent is a potential mitigating factor. Identification of the heterotrophic bacteria active in the denitrification process was attempted. This study presents a first report on the development and operation of the I-HRAP and has been followed by a technical-scale pilot plant evaluation of the process in the tertiary treatment of domestic wastewaters.

Saline deterioration in the South African public water system has been documented and disposal of brine wastes has been identified as part of the problem. The broad aim of this research programme was to undertake an initial technical study to evaluate the feasibility of integrating algal biotechnology into a disposal function for these wastes. A demonstration of utility in the form of products and waste treatment could produce a beneficiation of saline effluents and provide incentives necessary to deal with the disposal issue. The study attempted to demonstrate a synthesis between the two main thrusts in algal biotechnology that have produced large-scale practical applications - stable, predictable algal production in saline media and the cost effective High Rate Oxidation Ponding (HROP) process for incorporating algal production into a waste treatment function. Tannery organic saline effluents and the biotechnology of Dunaliella salina culture producing β- carotene were chosen as paradigms for the study. 1. The alga was shown to grow in certain tannery effluents producing enhanced biomass yields compared to defined inorganic medium cultivation. The potential for amino acid or protein supplementation of defmed culture media was noted. 2. A reduction in organic load simultaneous with the growth of D.salina was recorded in laboratory-scale simulations of the HROP process. Rates similar to the fresh water HROP equivalent were demonstrated. 3. These results suggested the uptake and storage of organic nitrogen by D.salina. The consequent inhibition of β-carotene accumulation by the organism presented a potentially insurmountable obstacle to the feasibility of β-carotene production in this medium. Uptake and release of organic compounds, previously demonstrated in phytoplankton and other micro-algae, was confirmed in this study for D.salina. The evidence acquired indicated the internalization of both glycine and bovine serum albumin. An ultrastructural study demonstrated mechanisms by which this process might occur. 4. The release of substantial quantities of glycerol was shown. A mechanism whereby D. salina may use this to regulate ammonia availability via control of its associated bacterial population was observed. Glycerol release was identified as presenting an application in treating refractory organic wastes, such as secondary sewage sludges, by elevating C:N ratios. This could demonstrate a significant utility for brine waste impoundments. 5. A multistage production process was proposed to deal with the problem of β-carotene inhibition by separation of the growth and metabolite accumulation functions into separate unit operations. It was shown in this study that the stress of nitrogen deficiency combined with high salinity provides for effectiveβ-carotene accumulation under the conditions of low illumination that pertain in dense cultures. Subjected to these conditions effluent-grown cells show delayed but unimpaired {j-carotene accumulation. 6. A role for the plant hormone abscisic acid in mediating the stress response was demonstrated in D.salina. Fluorescence induction studies suggested the presence of a signalling process forming part of a sensitivity control mechanism. Stress induction of β-carotene accumulation could occur through four clearly defined stages. Potential was identified for using this response as a physiological probe for monitoring and regulating the stress induction process. 7. The multistage processing concept requires effective algal cell separation technology. The use of cross-flow ultrafiltration and diafiltration with a polyethersulfone tubular membrane system was demonstrated as an effective process for the recovery and washing of D. salina. Cell concentrates were produced in a viable form. 8. Process designs incorporating the findings of the research programme are presented demonstrating how effluent and organic waste treatment functions may be combined with the production of D.salina and its products. Application of the multi-stage processing concept to β-carotene production in a defined medium process was identified as offering a potential four-fold yield enhancement. This could have a significant impact on a high cost, marginal algal biotechnology process. Aspects of novelty have been claimed in provisional patents applications. A provisional demonstration of the feasibility of D.salina production in tannery effluent indicates that algal biotechnology may provide a utility for, and hence the beneficiation of saline effluent wastes.

The biotechnology of production and utilisation of the cyanobacterium Spirulina has been well documented. Research has centred mainly on application in human and animal nutrition, and has been motivated by the high protein, vitamin, fatty acid and growth factor contents. The main obstacle in realising the full potential of this feed source has been the high production costs associated with its mass culture in defined media. The observation of blooms of Spirulina in tannery effluent evaporation ponds in Wellington, South Africa, prompted this investigation into the harvesting, and nutritional and toxicological evaluation of this potentially low-cost production system, with the ultimate aim of using the product in aquaculture rations. An investigation of the chemical gradient along the evaporation cascade showed a positive correlation between the prevailing chemical conditions and the dominant species populations. A standing crop of 9.5 tonnes/ha of Spirulina was found to be present in the latter alkaline ponds, characterised by relatively lower organic and sulphur contents. Initial harvesting of the biomass was achieved by the design, construction and implementation of a small-scale screen harvest, which yielded a 25 kg (dry weight) crop. A scale-up model was then designed, and implemented in a technical scale harvest, yielding a crop of 250 kg (dry weight). Both these harvests utilised the bloom of surface-autoflocculated biomass. Concentrated cell slurries were sun-dried on muslin beds, and milled to a coarse powder. An evaluation of the harvest revealed a chemical content similar to other published reports of defined media cultures, with the exception of the protein and amino acid contents. The observed lower levels of the latter two are almost certainly due to the sun-drying method employed, known to reduce the protein content due to thermal denaturation. Legislation demands the strict toxicological evaluation of new protein sources, and because of the effluent-nature of the growth medium of this source of Spirulina, its viability lies only in the application as an animal feed or supplement. A range of toxicological tests were chosen that were targeted to elucidate the possible toxicological constraints of this effluentgrown source of protein in animal nutrition. The nucleic acid and pesticide contents of the harvested biomass were within the prescribed safety ranges. Atomic absorption showed minimal accumulation of minerals and heavy metals from the effluent. A bioassay with the brine shrimp Anemia salina showed that the biomass contained no toxicologically active water-soluble components. A short term feeding trial with new-born chicks showed that supplementation with Spirulina had no effect on the growth rates and feed conversion ratios of the different feeding groups. Pathological analyses showed that the liver was the only target organ to elicit a change in response to supplementation of the diets with Spirulina. A general decrease in liver weight was noted, with Cu, Ca, Fe and Zn being significantly accumulated. A histopathological examination however, showed no cellular and functional aberration from the control animals. The toxicological analyses gave the preliminary safe go-ahead for the evaluation of effluent-grown Spirulina in aquaculture nutrition. The South African abalone Haliotis midae, and the rainbow trout Oncorhynchus mykiss were chosen as representative species of edible cultured organisms. The technology for the culture of the perlemoen abalone is being established in South Africa, with the main area of research being the development of an artificial diet for high density culture. A 40 day growth trial demonstrated that lower concentrations of Spirulina supplemented to an agar-based fishmeal diet resulted in growth rates and feed conversion ratios similar to the control fishmeal and purified-casein diets, and thus has application potential in the nutrition of this high-cost marine delicacy. The aquaculture technology of freshwater rainbow trout is already well established. An eight week feeding trial with various concentrations of Spirulina showed that this effluent-grown protein source can partially replace fishmeal in semi-purified diets. Fish fed Spirulina did not exhibit decisive manifestations of toxicity, as determined in a histopathological study. In addition, Spirulina supplementation resulted in enhanced colouration of the skin and flesh, which may have implications in the aesthetic marketing of this sought-after table fish. The primary aim of this preliminary investigation thus concerned the determination of the biotechnological potential of this effluent-source of Spirulina. A technology transfer from the economically unfeasible defined-media culture was implemented. This project is ultimately aimed as a contribution towards the treatment of tannery wastewater, by the removal of contaminants from the effluent in the form of organic biomass.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Breast cancer remains the leading cause of cancer-related death in women worldwide. Furthermore, it has been demonstrated that the treatment-resistant ER-PR-HER2/neu- sub-type is more common among women of African descent, necessitating the search for novel chemotherapies for this form of the disease. The secondary metabolites produced by marine algae represent a rich source of structurally unique compounds with chemotherapeutic potential, particularly in South Africa, whose oceans allegedly host 15 % of the total number of species in the world. Indeed, a recent study reported the isolation of a range of novel compounds from South African red and brown algae of the Plocamium, Portiera and Sargassum genera which displayed cytotoxicity against oesophageal cancer cells in vitro. The molecular mechanisms mediating this toxicity were unknown, as was the effect of these and similar compounds on metastatic ER-PR-HER2/neu- breast cancer cell lines or breast cancer stem cells. The current study aimed to address these questions by screening a library of twenty-two novel marine algal compounds for the ability to inhibit MDA-MB-231 and Hs578T breast cancer cells, while having no adverse effects on non-cancerous MCF12A breast epithelial cells. While twelve of these were toxic in the micromolar range against breast cancer cells, only the polyhalogenated monoterpenes RU004 and RU007, and the tetraprenylated quinone sargaquinoic acid (SQA) were identified as hit compounds based on the criteria that their cytotoxicity was specific to breast cancer and not healthy breast cells in vitro. On the other hand, the halogenated monoterpene RU015 was found to be highly toxic to both breast cancer and non-cancerous breast cell lines, while the halogenated monoterpene stereoisomers RU017 and RU018 were non-toxic to either of these cell lines. The mode of action of RU004, RU007, RU015 and SQA, together with the previously characterized carotenoid fucoxanthin (FXN), was assessed in terms of the type of cell death induced and the effect on cell cycle distribution of these compounds. Flow cytometric analysis of the extent of Hoescht 33342 and propidium iodide staining along with PARP cleavage studies suggested that SQA induced apoptosis in MDA-MB-231 cells. On the other hand, the highly toxic compound RU015 appeared to induce necrosis as evidenced by 50 kDa PARP cleavage product in MDA-MB-231 cells. The flow cytometry profiles of MDA-MB-231 and Hst578T cells treated with the hit compounds RU004 and RU007 were suggestive of the induction of apoptosis by these compounds. Cell cycle analysis by flow cytometry with propidium iodide staining revealed that both SQA and FXN induced G0-G1 arrest together with an increase in the apoptotic sub-G0 population, which agreed with previous reports in the literature. The molecular mechanism of action of SQA and FXN were further investigated by the identification of specific signal transducer molecules involved in mediating their anti-cancer activities. SQA was found to require the activity of numerous caspases, including caspase-3, -6, -8, -9, -10 and -13, for its cytotoxicity and was demonstrated to decrease the level of the antiapoptotic protein Bcl-2. On the other hand, FXN was shown to require caspase-1, -2, -3, -9 and - 10 for its toxicity. This, together with the ability to decrease the levels of Bcl-2, pointed to the involvement of the intrinsic pathway in particular in mediating the activity of FXN. The screening of algal compounds against non-cancerous breast epithelial cells carried out in this study, together with the investigation into their mechanisms of action, represent one of the few reports in which characterization of algal metabolites goes beyond the initial cytotoxicity assays. Finally, in order to assess the potential anti-cancer stem cell activity of the marine algal compounds, a subset of these was screened using a mammosphere assay technique developed in this study. The cancer stem cell (CSC) theory proposes that cancers arise from and are maintained by a specific subpopulation of cells able to undergo asymmetric cell division and termed CSCs. These CSCs are capable of anchorage-independent growth in serum-free culture conditions, such as those in the mammosphere assay. Using this assay, the novel halogenated monoterpene stereoisomers RU017 and RU018 were demonstrated to possess putative anti- CSC activity as evidenced by their ability to completely eliminate mammosphere formation in vitro. Furthermore, since RU017 and RU018 were non-toxic to both breast cancer and healthy breast cells, it appeared that the activity of the compounds was potentially specific to the CSCs. The results require further validation, but represent the first report of selective anti-CSC activity.

This study investigated the possible use of algal extracellular polysaccharide as a biosorbent for removal of heavy metals (copper and lead) from aqueous solutions as a means of bioremediation for metal containing effluents. This biopolymer has good biosorbent properties and a potential to provide a cost effective, selective and efficient purification system. A variety of environmental conditions induce the production of extracellular polysaccharides in algae. The production of exopolysaccharides by Dunaliella cultures was induced by nitrogen deficient conditions. A high ratio of carbon to nitrogen source considerably enhanced the polysaccharide release. Purified extracellular polysaccharide samples exhibited a monosaccharide composition consisting of the following sugars: xylose, arabinose, 2-0-methyl mannose, mannose, glucose and galactose. The relative abundance (%) of these sugars were calculated relative to xylose. The major sugar constituent was 2-0-methyl mannose, which was present at approximately 160% relative to xylose. The percentage relative abundance of other sugars was as follows: 18.8; 86.8; 85.3 and 22.3% for arabinose; mannose; glucose and galactose respectively. The identity of the various constituents were confirmed by mass spectrometry. The ability of Dunaliella exopolysaccharides to accumulate metals was investigated. The following parameters were studied because they affect metal uptake: solution pH, biomass concentration, temperature, time and metal concentration. The uptake of both copper and lead were pH dependent. However, metal uptake was not significantly affected by temperature. Kinetic studies showed that Dunaliella extracellular polysaccharides exhibit good bioremediation properties. Metal uptake was rapid. In addition, the exopolysaccharide has good metal binding capacity with an uptake capacity for lead of 80 mg/g from a solution containing initial lead concentration of approximately 40 mg/l. Competition studies revealed that the presence of a second metal in solution inhibits uptake of the other metal compared to uptake in single metal solution of that particular metal. The presence of lead inhibited the uptake of copper from approximately 65% in single metal solution to 10% in binary metal solution. The presence of copper also inhibited lead uptake, though not to the same extent. Higher concentrations of lead could not completely prevent removal of copper from solution and visa versa. The same was true for lead which could not be displaced by a four-fold concentration of copper. Instead, a certain percentage of copper was always removed showing that lead did not compete with copper for these binding sites. In conclusion it appears that, copper and lead bind to different sites on Dunaliella exopolysaccharides and that they exhibit selective or preferential removal of lead.