Made available in DSpace on 2014-06-11T19:23:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-25Bitstream added on 2014-06-13T19:49:36Z : No. of bitstreams: 1 crulhas_bp_me_rcla_parcial.pdf: 100924 bytes, checksum: c0953845b12fd7e562861860ab7b97ca (MD5) Bitstreams deleted on 2015-06-25T13:01:17Z: crulhas_bp_me_rcla_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:31Z : No. of bitstreams: 1 000713376_20160402.pdf: 87205 bytes, checksum: b056e25228f33bc3a3f95fc8c1ac65c2 (MD5) Bitstreams deleted on 2016-04-04T12:10:27Z: 000713376_20160402.pdf,. Added 1 bitstream(s) on 2016-04-04T12:11:07Z : No. of bitstreams: 1 000713376.pdf: 656987 bytes, checksum: f6bb8af3851049442710d3f18ac08f28 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A luz incidente é certamente uma das principais variáveis ambientais que influenciam a distribuição, estrutura e funcionamento das comunidades de macroalgas de ecossistemas lóticos. Por isso, a influência da variação qualitativa e quantitativa da luz incidente sobre as respostas fotossintéticas foi analisada em onze táxons macroalgais pertencentes aos Filos Chlorophyta, Cyanobacteria, Rhodophyta e Xanthophyta, utilizando as técnicas de oxigênio dissolvido e fluorescência da clorofila a. Através destas técnicas foram produzidas curvas de fotossíntese-irradiância e então calculados vários parâmetros fotossintéticos. As espécies do Filo Chlorophyta registraram uma preferência pela luz verde, com altos valores de Pmax, eficiência fotossintética e rendimento quântico efetivo, nesta faixa do espectro luminoso. Em relação à quantidade de luz disponível, o grupo apresentou padrões espécie-específicos, tanto com espécies tidas como de sombra como de sol. O Filo Cyanobacteria apresentou melhor aproveitamento fotossintético quando exposto à luz vermelha, pois, obteve altos valores de rendimento quântico efetivo e eficiência fotossintética neste tratamento. Quanto à quantidade de luz incidente, o grupo demonstrou resposta indiferente, sem uma clara tendência nos seus parâmetros fotossintéticos nos tratamentos de atenuação. O Filo Rhodophyta demonstrou altos valores de eficiência fotossintética e rendimento quântico efetivo nos espectros luminosos azul e vermelho e, considerando os dados quantitativos, o grupo apresentou respostas fotossintéticas típicas de plantas de sombra, com baixos valores de Pmax, e do ponto de saturação e altos valores de rendimento quântico efetivo e eficiência fotossintética sob menores irradiâncias. O Filo Xanthophyta, no experimento qualitativo, obteve... / The incident light is certainly one of the main environmental variables which influence the distribution, structure and functioning of lotic macroalgae communities. Therefore, the influence of qualitative and quantitative variation of incident light on the photosynthetic responses were analyzed in eleven especies pertaining to Chlorophyta, Cyanobacteria, Rhodophyta and Xanthophyta, using the techniques of dissolved oxygen and chlorophyll a fluorescence. Through these techniques the irradiance curves were made and then the photosynthetic parameters were analyzed. The species of Chlorophyta recorded a preference for the green light, with high Pmax, photosynthetic efficiency and quantum yield, in this range of the light spectrum. Relative to the quantity of light available, the group presented speciesespecific patterns with both sun and shade species. The Cyanobacteria showed better exploitations when exposed to red light, whereas, obtained high values of effective quantum yield and photosynthetic efficiency in this treatment. Regarding to the amount of incident light, the group showed indifferent response, with no clear trend in its photosynthetic parameters in the attenuation treatments. The Rhodophyta, in the qualitative experiment demonstrated high values of photosynthetic efficiency and effective quantum yield in the blue and red spectrum, and in the quantitative experiment, the group presented photosynthetic results typical of shade plants due to low Pmax and saturation point, and high values of effective quantum yield and photosynthetic efficiency under lower irradiances. The Xanthophyta obtained milder and similar values of photosynthetic parameters among the tested treatments in the qualitative experiment, being indifferent to the type of available spectrum. In the quantitative experiment, it showed a preference... (Complete abstract click electronic access below)

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Arsenic (As) exposure through water and As contaminated food in rural areas around the world is well documented. While there are accurate, precise, and even robust screening methods for on-site water analysis, the determination of toxic inorganic As (iAs, a class I carcinogen) in foodstuff has been made possible through methods based on mass spectrometry. No screening or field method for iAs in food has been established and, there is also a lack of screening and monitoring methods for human exposure to iAs. The objectives of this thesis were to develop and apply a robust, reliable and well established screening method which is field deployable for the measurement of iAs in rice and seaweed in addition to the total As metabolites in human urine resulting from exposure to inorganic As. Reported in this work is the development and application of optimised field deployable methods based on the Gutzeit reaction with the aid of a field test kit (FTK) for the determination of iAs in rice, rice-based products, edible seaweeds and seaweeds cultivated from their natural habitat. The methods involve simple sample extraction by boiling in nitric acid before analysis with the FTK. Results were obtained in under an hour with the FTK and further validated with speciation analysis by HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry). Analysis of 30 store-bought rice samples with the field method gave good reproducibility (± 12 %) for samples with variable As concentrations. The results were comparable to those obtained by HPLC-ICP-MS with no contribution from organoarsenicals. Screening analysis with the field method based on recent regulations for inorganic arsenic in rice also gave low false positive and false negative rates ( < 10 %) for violations against these regulations, an indication that the method can accurately identify samples that are above or below the recommended maximum contaminant limits for iAs in rice. Similarly, results from the seaweed analysis with the field method were also comparable to those from speciation analysis by HPLC-ICP-MS with limited bias between the set of data from both vii methods. Optimisation of extraction methods using a subset of samples gave 80-95% iAs recovery with no contribution from the organoarsenicals present in the samples. The determination of total As metabolites in urine from the exposure to iAs could not be done directly using the FTK. In this case, the method involved the use of UV photolysis with persulphate and titanium dioxide as oxidizing agents for the conversion of methylated As species (DMA) to the inorganic form before analysis with the FTK. A partial determination of DMA with the FTK in urine matrix was demonstrated but this needs to be studied further for the development of a robust field method for monitoring human exposure to iAs.

Background: The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sister to more complex multicellular volvocine families, Goniaceae and Volvocaceae. Thus, T. socialis is a key species to elucidate the initial steps in the evolution of multicellularity. In the asexual life cycle of C. reinhardtii and multicellular volvocine species, reproductive cells form daughter cells/colonies by multiple fission. In embryogenesis of the multicellular species, daughter protoplasts are connected to one another by cytoplasmic bridges formed by incomplete cytokinesis during multiple fission. These bridges are important for arranging the daughter protoplasts in appropriate positions such that species-specific integrated multicellular individuals are shaped. Detailed comparative studies of cytokinesis between unicellular and simple multicellular volvocine species will help to elucidate the emergence of multicellularity from the unicellular ancestor. However, the cytokinesis-related genes between closely related unicellular and multicellular species have not been subjected to a comparative analysis. Results: Here we focused on dynamin-related protein 1 (DRP1), which is known for its role in cytokinesis in land plants. Immunofluorescence microscopy using an antibody against T. socialis DRP1 revealed that volvocine DRP1 was localized to division planes during cytokinesis in unicellular C. reinhardtii and two simple multicellular volvocine species T. socialis and Gonium pectorale. DRP1 signals were mainly observed in the newly formed division planes of unicellular C. reinhardtii during multiple fission, whereas in multicellular T. socialis and G. pectorale, DRP1 signals were observed in all division planes during embryogenesis. Conclusions: These results indicate that the molecular mechanisms of cytokinesis may be different in unicellular and multicellular volvocine algae. The localization of DRP1 during multiple fission might have been modified in the common ancestor of multicellular volvocine algae. This modification may have been essential for the re-orientation of cells and shaping colonies during the emergence of multicellularity in this lineage.

Integrated algae pond systems (IAPS) combine the use of anaerobic and aerobic bioprocesses to effect wastewater treatment. Although, IAPS as a technology process offers many advantages including efficient and simultaneous N and P removal, no requirement for additional chemicals, O2 generation, CO2 mitigation, and a biomass with potential for valorization, a lack of technological advancement and the need for large land area, has limited the reach of this technology at industrial scale. In mitigation, peroxonation was introduced as a tertiary treatment unit and its effect on COD and TSS of IAPS treated water investigated. An effort was made to characterize the soluble but persistent COD in IAPS treated water and, productivity of the HRAOP mixed liquor was investigated to gain insight into the potential use of this biomass. Results show that peroxone treatment effectively reduced COD, TSS, and nutrient load of IAPS water without any significant impact on land area requirement. Indeed, summary data describing the effect of peroxone on quality of IAPS-treated water confirmed that it complies with the general limit values for either irrigation or discharge into a water resource that is not a listed water resource for volumes up to 2 ML of treated wastewater on any given day. Extraction followed by FT-IR spectroscopy was used to confirm albeit tentatively, the identity of the soluble but persistent COD in IAPS treated water as MaB-floc EPS. Results show that MaB-flocs from HRAOPs are assemblages of microorganisms produced as discrete aggregates as a result of microbial EPS production. A relationship between photosynthesis and EPS production was established by quantification of the EPS following exposure of MaB-flocs to either continuous light or darkness. Several novel strains of bacteria were isolated from HRAOP mixed liquor and 16S ribosomal genomic sequence analysis resulted in the molecular characterization of Planococcus maitriensis strain ECCN 45b. This is the first report of Planococcus maitriensis from a wastewater treatment process. Productivity and change in MaB-flocs concentration, measured as mixed liquor suspended solids (MLSS) between morning and evening were monitored and revealed that MLSS is composed of microalgae and bacteria but not fungi. Concentration varied from 77 mg L-1 in September (winter) to 285 mg L-1 in November (spring); pond productivity increased from 5.8 g m-2 d-1 (winter) to 21.5 g m-2 d-1 (spring); and, irrespective of MLSS concentration in late afternoon, approximately 39% was lost overnight, which presumably occurred due to passive removal by the algae settling pond. The outcomes of this research are discussed in terms of the quality of treated water, and the further development of IAPS as a platform technology for establishing a biorefinery within the wastewater treatment sector.

Philosophiae Doctor - PhD / Recently, a great deal of interest has developed towards the isolation of bioactive compounds from marine sources due to their numerous health benefits. Furthermore, marine algae are valuable sources of structurally diverse metabolites with scientifically proven therapeutic claims. The cell walls are rich in sulfated polysaccharides such as fucoidans in brown algae, carrageenans in red algae and ulvans in green algae. These sulfated polysaccharides exhibit many beneficial biological activities such as anticoagulant, antiviral, antioxidative, anticancer and immunomodulating activities. They have great potential for further development as products in cosmeceutical, pharmaceutical and nutraceutical areas. Although the mechanism of action is still not clear, their biological activities could be mainly attributed to their major secondary metabolites namely; phlorotannins, terpenoids and fucoidans. There was use of comprehensive chromatographic separations and a full analysis of isolates using one or other of the spectroscopic techniques. Antioxidant and cytotoxicity tests were perfomed in details for Ecklonia maxima. Furthermore, structural and electronic features of the phlorotannins were compared in an attempt to provide an explanation for the differences in their radical scavenging properties. In this regard, two main radical scavenging mechanisms, hydrogen atom transfer (HAT) and electron transfer (ET), were assessed in order to determine the preferred mode of radical scavenging. Fully relaxed geometry optimizations of the neutral and the radical species were performed utilizing DFT/B3LYP and DFT/UB3LYP methods respectively. In further studies, the structural and functional properties of sulfated polysaccharides from the three brown and one red seaweeds were investigated. This was through detailed analysis of chemical composition of crude and purified polysaccharides using PMP - derivatization of hydrolysed sugars, anion exchange, molecular weight determination, ion chromatography , FT-IR, NMR to methylation analysis. The work reports isolation and characterization of compounds from four algae: Ecklonia maxima, phlorotannin derivatives, namely phloroglucinol (22), eckol (23), 7-phloroeckol (24), 2-phloroeckol (25) and a sterol, 24-ethylidine cholesterol (26); Splachinidium rugosum, 24-ethylidine cholesterol ( 26), 1, 3-Dicapryloyl-2-oleoylglycerol (27), E-3,7,11,15-tetramethylhexa dec-2-en-1-ol (phytol) (2 8); Macrocystis angustifolia, 24-ethylidine cholesterol (26); a red seaweed Aeodes orbitosa, and E -3, 7, 11, 15-tetramethylhexad ec-2-en-1-ol (28) and 17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7, 8,9,11,12,14,15,16,17-dodecahydro-1 H-cyclopenta[a]phenanthren-3-ol (β-sitosterol) (29). Experimental findings and theoretical predictions of phlorotannins indicated that the radical scavenging activities followed the order 22< 23 < 25 < 24. Theoretical studies further indicated the ET mechanism is more significant than the HAT mechanism due to the high BDE values. Their polysaccharide structures were tentatively shown to have a backbone of (1-3) and (1-4) linkages with sulfate groups at O-2 and O-2, 3 positions. The only red algae studied contained, 2-O-methyl-D-galactose with (1-3) and (1-4)-glycosidic linkages possessing sulfate groups at positions 2 and 6.

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.

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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.

During the evolution of multicellularity from unicellular ancestors, cells transition from being evolutionary individuals to components of more complex, multicellular evolutionary individuals. The volvocine green algae provide a powerful model system for understanding the genetic and morphological changes that underlie and are caused by the evolution of multicellularity. This dissertation concerns the role of cell cycle regulation, cellular differentiation, and sexual traits during the evolution of multicellularity. While some of these are shown to be causally important in the origins of multicellularity (Appendix B), others are driven by the evolution of multicellularity (Appendix D). We provide a review of recent mathematical models on the evolution of multicellularity, which are found to focus heavily on the later, subsequent stages of the evolution of multicellular complexity. We found that many of these models assume multicellular ancestors and instead evolve cellular differentiation, bringing attention to a gap in our understanding of the events in the initial stages of the evolution of multicellularity. We show that a focus on the early stages of the evolution of multicellularity reveals a powerful and critical role for regulation of the cell cycle at the origins of multicellularity (Appendix B). We further find that the genetic basis for cellular differentiation evolved sometime after the evolution of cell cycle regulation. We find that while the genetic basis for cellular differentiation evolved after cell cycle regulation, it also evolved earlier than previously predicted in the volvocine green algae, suggesting an important role in undifferentiated species (Appendix C). Lastly, having elucidated the origins and evolution of multicellularity, we find that multicellularity causes the evolution of sexual traits including anisogamy, internal fertilization, and subsequently sexual dimorphism (Appendix D). This work emphasizes the important role that multicellularity plays in driving the evolution of sexual diversity seen across the eukaryotic tree and well as informs critical hypotheses on the evolution of anisogamous sex, among the most challenging problems in evolutionary theory.