M. Tech (Biosciences, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The mass production of algae for commercial purposes has predominately been carried out in open ponds systems. However, open ponds systems have a number of disadvantages such as poor light utilization, requirement for large areas of land and high risks of contamination. On the other hand, photobioreactors have attracted much interest because they allow a better control of the cultivation conditions than open systems. With photobioreactors, higher biomass productivities are obtained and contamination can be easily prevented. Photobioreactors can also be engineered to manipulate the light and dark photosynthetic reactions thus enhancing biomass productivity. The main objective of this study was to construct a novel tubular photobioreactor which had the ability to expose the cultured alga to light and dark phases with the aim of optimizing the algal biomass production. A novel tubular photobioreactor with the ability to manipulate the cultured alga’s light and dark photosynthetic reactions was constructed in this study. The alga Spirulina platensis was chosen as the test organism in this novel tubular photobioreactor due to a number of reasons such as its globally socioeconomic importance, its tolerance of higher pH and temperature values which makes it almost impossible to contaminate. The cultivation process of Spirulina in the photobioreactor was investigated through alternating light and dark cycles in an attempt to increase the photosynthetic efficiency of the culture. The effect of different light intensities on the growth of Spirulina in the novel tubular photobioreactor was investigated and it was found that the best light condition that favored higher biomass formation was at 600 μ mol m-2 s-1. Five different light/ dark ratios were evaluated at a light intensity of 600 μ mol m-2 s-1 during a batch mode of operation of the novel tubular photobioreactor. The light/ dark ratio of 1:0.25 was found to be the best ratio because it gave the highest biomass in the shortest period of time when compared to the other ratios used. These results seem to suggest that longer light cycle relative to dark cycle results in higher biomass production. The ratio of 1:0.25 was then used to operate the novel tubular photobioreactor in a continuous mode. A maximum biomass productivity of 25 g/m2/day was achieved which corresponded to a net photosynthetic efficiency of 5.7 %. This result was found to be higher than what most photobioreactors could achieve but it was 2.8 g/m2/day lower than the highest ever reported productivity in a photobioreactor when Spirulina is cultivated. The 2.8 g/m2/day lower was attributed to the different materials used in the construction of these two photobioreactors. The photobioreactor which achieved 27.8 g/m2/day was made up of a clear glass whereas the novel tubular photobioreactor was made up of a PVC tubing. PVC tubes tend to change from clear to a milky colour after a certain period when it is used at higher temperature and pH values hence blocks a certain amount of light. Therefore the main recommendation in this study is to use a PVC tubing with a longer life span when used at a higher temperature and pH values.

The aim of this dissertation was to elaborate the exploration of biologically active secondary metabolites from the marine sponge Cacospongia cf. linteiformis collected from the Bahamas and the soft coral Briareum asbestinum collected from two different sites in Florida State, Boca Raton and Dry Tortugas. In chapter one, a review on previous chemical and biological studies of the marine sponge C. cf. linteiformis and soft coral B. asbestinum is provided. Particular attention is given to spongianolides and briarellins, two important classes of compounds isolated from C. cf. linteiformis and B. asbestinum, respectively, and their structural features and diverse bioactivities. In chapter two, the isolation and relative configuration determination of four epimeric sesterterpenoids, spongianolides E & F (18c, 18d, 19c, 19d) from C. cf. linteiformis collected from the Bahamas are discussed. Thanks to chemical modification (acetylation), diastereomeric 18c&18d and 19c&19d, respectively, were able to be isolated using chromatographic techniques for the first time, and then the relative configurations of 18c, 18d, 19c, 19d were determined based on NOESY NMR experiments. The bioactivity of mixture of compounds 18c, 18d, 19c, 19d were tested and it exhibited inhibition against Schnurri-3 (a regulator of postnatal bone mass). In chapter three, the isolation and structural elucidation of four new compounds, florellins A-D (49-52), from B. asbestinum collected off the coast of Boca Raton, FL are discussed. The molecular structures of these compounds were established by spectroscopic analysis. Compounds 49-52 are the first briarellins containing an acyl group at C-13, while 49 and 50 are the first briarellins possessing acylation at C-15. Florellins A–C (49-51) were screened and found cytotoxic against three human cell lines, BT474, WM266−4 and HEK293. In chapter four, the isolation and structural elucidation of four new compounds, florellins E-H (57-60), from B. asbestinum collected in Dry Tortugas, FL are discussed. The molecular structures of these compounds were established by spectroscopic analysis. Florellins F (58) and H (60) were screened against three human cell lines, BT474, WM266−4 and HEK293, but no cytotoxicity was exhibited. In chapter five, all the experimental procedures are described, including analytical instruments, animal materials, extraction and isolation processes, spectroscopic data and protocols of bioassays. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

The application of high rate algal ponds (HRAP) in the treatment of brewery effluent that met the South African Department of Water Affairs and Forestry's (DWAF) general limits for discharge into a natural water resource of 1998 were tested during a lO-month baseline phase, followed by an 11-month optimization phase. The objective of the baseline phase was to monitor the seasonal performance of HRAPs. The hydraulic retention time (HRT) fluctuated between 11.16 d and 12.00 d in HRAPs. The chemical oxygen demand (COD) increased from 130.12 ± 6.94 mg/L (post-AD), to 171.21 ± 7.99 mg/L (post-HRAP) . The presence of algal cells and evaporation contributed towards an increase in post-HRAP COD. The ammonia (NH₄-N) concentration decreased from 46.59 ± 2.47 mg/L (post-AD), to 1.08 ± 0.12 mg/L (post-HRAP). The nitrite (NO₂- N) concentration remained below 1.00 mg/L in post-pilot plant AD, post-PFP and post-HRAP effluent. The phosphate (PO₄-P) concentration decreased from 29.81 ± 1.39 mg/L (post-AD) to 17.30 ± 1.16 mg/L PO₄-P. The objective of the optimization phase was to manipulate the HRT to achieve the maximum treatment rate that met the DWAF general limits for discharge into a natural water resource of 1998. Nitrogen (as NH₄-N, NO₃-N, NO₂-N) removal efficiency was used as an indicator of nutrient removal success. HRT was influenced by season. The optimal HRT for autumn was 4.30 d at a temperature of 20.53ºC in HRAP A2 (heated) and 18.96ºC in HRAP B2 (ambient). The optimal HRT for summer was 2.74 d at 29.90ºC in HRAP A2 (heated) and 26.36ºC in HRAP B2 (ambient). The COD decreased from 152.33 ± 4.85 mg/L (post-AD) to 95 .00 ± 3.75 mg/L (post-HRAP A2), and to 100.82 ± 5.93 mg/L (post-HRAP B2). The incoming NH₄-N concentration decreased from 42.53 ± 1.38 mg/ L (post-AD), to 1.70 ± 0.81 mg/ L (post-HRAP) . The nitrate (NO₃-N) concentration post-HRAP was 12 - 14 mg/L. The main methods for NH₄-N removal were probably NH₄-N volatilization through algal uptake. HRAPs were able to lower nitrogen and phosphorous concentrations to within the DWAF limits under normal operating conditions. It is recommended that HRAP treated brewery wastewater be used for irrigation after salt removal, or alternatively, for groundwater recharge . Regulatory exemptions would be required for higher than permitted COD and EC concentrations to enable these actions.

Brewery effluent that had undergone treatment in an anaerobic digester (AD) was used as an alternative water and nutrient source for hydroponic crop production. Brewery effluent was demonstrated to contain sufficient nutrients to support the growth, flowering and fruiting of Lycopersicum escolentum "Moneymaker" tomato crops. The adjustment of the effluent pH with phosphoric acid to between pH 6.0 and 6.5 increased the development of the crops by around 100% compared to crops grown in unaltered effluent. The pH adjusted effluent-grown plants grew to a mean height of 831.4 ± 21.1 mm and a dry biomass weight of 42.34 ± 2.76 g compared to the unaltered pH effluent plants which grew to a height of 410.6 ± 20.5 mm and a weight of 7.65 ± 0.68 g after 49 days. Effluent treatment in high-rate algal ponds (HRAP) was determined to have no positive effect on the nutritional potential of the effluent for Moneymaker production. The effluent-grown plants did not perform as well as plants grown in inorganic-fertilizer and municipal water. Plants grown in effluent grew taller but did not produce significantly more fruit when phosphoric acid (height: 1573.3 ± 50.4 mm, 19.4 ± 1.4 fruit per plant) was compared to nitric acid (height: 1254.1 ± 25.4 mm, 15.6 ± 1.5 fruit per plant) as the pH adjustment over 72 days. Direct and secondary plant stresses from effluent alkalinity, ammonium nutrition, nitrogen limitation, sodium concentrations and heat stress among other factors were probably confounding variables in these trials and require further investigation. Considering the raw effluent composition and manipulating the AD operation is a potential opportunity to improve overall AD performance, reduce chemical inputs in the effluent treatment process, reduce the final effluent alkalinity, and increase available nitrogen content in the final effluent. The anaerobic digester discharging >1000 m³ of nutrient enriched effluent every day is a resource with considerable potential. The benefits of developing this resource can contribute to cost-reduction at the brewery, more efficient water, nutrient and energy management at the brewery, and offer opportunities for job creation and potentially benefit local food security.

Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.

High rate algae oxidation ponds (HRAOP) for domestic wastewater treatment generate biosolids that are predominantly microalgae. Consequently, HRAOP biosolids are enriched with minerals, amino acids, nutrients and possibly contain plant growth regulator (PGR)-like substances, which makes HRAOP biosolids attractive as fertiliser or PGR. This study investigated HRAOP biosolids as a starting material for a natural, cost-effective and readily-available eco-friendly organic fertiliser and/or PGRs. Various HRAOP extract formulations were prepared and their effect on plant growth and development was evaluated using selected bioassays. Initial screening included assessing the effect on change in specific leaf area, radish cotyledon expansion as an indicator of PGR-like activity, and seed germination index (GI). More detailed studies on fertiliser efficacy and PGR-like activity utilised bean (Phaseolus vulgaris) and tomato (Solanum lycopersicum) plants. Combined effects of sonicated (S) and 40% v/v methanol (M) extract (5:1 SM) had impressive plant responses, comparable to Hoagland solution (HS). Other potentially fertiliser formulations included 0.5% M, 1% M, 2.5% S and 5% S formulations. The 5:1 SM and 5% S showed greater PGR-like activity, promoting cotyledon expansion by 459 ± 0.02% and 362 ± 0.01%, respectively. GI data showed that none of the formulations negatively impacted germination. Further investigation showed that the 5% S formulation increased leaf length, width and area by 6.69 ± 0.24, 6.21 ± 0.2 mm and 41.55 ± 0.2 mm². All formulated fertiliser extracts had no adverse effect on chlorophyll content and plant nutrient balance as indicated by C:N (8-10:1) ratio. In addition, plants appeared to actively mobilise nutrients to regions where needed as evidenced by a shift in shoot: root ratio depending on C, N and water availability. Furthermore, 5% S caused a 75% increase in tomato productivity and had no effect on bean productivity. Whereas, 5:1 SM and 1% M formulation improved bean pod production by 33.3% and 11%, respectively but did not affect tomato production. Harvest index (HI) however indicated a 3% reduction in tomato productivity with 5:1 SM and little or no enhancement in bean productivity with both 5:1 SM and 5% S treatments. Bean plants treated with 5:1 SM and 5% S produced larger fruits, which could be an indication of the presence of a PGR effect. Overall, HRAOP biosolids extracts prepared and investigated in this study demonstrated both fertiliser characteristics and PGR-like activity with performances comparable and in some cases exceeding that of commercial products. However additional research is needed to confirm presence of PGR-like activities and fertiliser efficacy.

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.

Integrated Algae Pond Systems (IAPS) are a derivation of the Oswald designed Algal Integrated Wastewater Pond Systems (AIWPS®) and combine the use of anaerobic and aerobic bioprocesses to effect wastewater treatment. IAPS technology was introduced to South Africa in 1996 and a pilot plant designed and commissioned at the Belmont Valley WWTW in Grahamstown. The system has been in continual use since implementation and affords a secondarily treated water for reclamation according to its design specifications which most closely resemble those of the AIWPS® Advanced Secondary Process developed by Oswald. As a consequence, and as might be expected, while the technology performed well and delivered a final effluent superior to most pond systems deployed in South Africa it was unable to meet The Department of Water Affairs General Standard for nutrient removal and effluent discharge. The work described in this thesis involved the design, construction, and evaluation of several tertiary treatment units (TTU') for incorporation into the IAPS process design. Included were; Maturation Ponds (MP), Slow Sand Filter (SSF) and Rock Filters (RF). Three MP's were constructed in series with a 12 day retention time and operated in parallel with a two-layered SSF and a three-stage RF. Water quality of the effluent emerging from each of these TTU's was monitored over a 10 month period. Significant decreases in the chemical oxygen demand (COD), ammonium-N, phosphate-P, nitrate-N, faecal coliforms (FC) and total coliforms (TC) were achieved by these TTU's. On average, throughout the testing period, water quality was within the statutory limit for discharge to a water course that is not a listed water course, with the exception of the total suspended solids (TSS). The RF was determined as the most suitable TTU for commercial use due to production of a better quality water, smaller footprint, lower construction costs and less maintenance required. From the results of this investigation it is concluded that commercial deployment of IAPS for the treatment of municipal sewage requires the inclusion of a suitable TTU. Furthermore, and based on the findings presented, RF appears most appropriate to ensure that quality of the final effluent meets the standard for discharge.