Philosophiae Doctor - PhD / In South Africa the green macroalga Ulva armoricana is the main species of macroalgae cultured. The species is currently the largest aquaculture (2884.61 tonnes) product by weight with a corresponding capacity for biogas (CH₄) production. We have shown that biotransformation of U. armoricana to Liquefied Petroleum Gas (LPG) is viable and economically feasible as a clean fuel. pH toxicity tests showed that U. armoricana can be used as a health index, under potentially increased CO₂ concentrations that can occur in IMTA carbon sequestration. We have shown sporulation to be the morphological response to environmental stress, which is indicative of chlorophyll degradation and a reduction in the photosynthetic activity of the alga. With the exception of Cadmium (Cd), the physico-chemical values obtained and the dissolved nutrient/heavy metals uptake by the alga all fell within the FAO/WHO permissible standards. Our Cd values therefore negate the use of these macroalgae for human consumption. We have also shown that U. armoricana can be used in eco-monitoring by playing a significant role in wastewater filtration and bioaccumulation. Nutrient utilization and proximate composition results show that African mud catfish (Clarias gariepinus) grow well on a protein-enriched Ulva diet, suggesting that enriched Ulva has the potential to be a successful fish feed. This thesis suggests among others, that South Africa could take advantage by being the first African country to propose specific standards for edible macroalgae as its successful research innovations and development provides a template for other African countries to further their aquaculture sectors. Additional benefits (bioremediation, ocean de-acidification through the capture of atmospheric and dissolved CO₂ during growth to assist in climate change mitigation) from Ulva farming activities bode well for the aquaculture industry.

Seaweeds are among the five marine sub-sectors of species cultured in South Africa, with Ulva species cultured at a commercial scale. In South Africa, the annual production of Ulva is approximately 2000 tonnes (wet weight), with the majority of Ulva being grown in landbased paddle raceway systems receiving abalone effluent water. Cultured Ulva is mainly used as abalone feed and for bioremediation of farm effluent water. It is not sold but rather is used either as fresh feed or dried and incorporated into formulated feeds. The main commercial abalone farms growing Ulva in paddle raceway systems in South Africa are Irvin & Johnson (I&J) Cape Abalone, Abagold, Buffeljags Abalone and Diamond Coast Aquaculture in the Western Cape province, and Wild Coast Abalone in the Eastern Cape province. The main aim of this study is to precisely identify the cultured Ulva species as their identity is not clearly understood. This will provide information on the genetic diversity in the cultured material and could enable farmers to select for a species/strain that has the desired traits such as high nutritional value, rapid growth rate, resistance to diseases, and the ability to grow vegetatively, amongst others, so that the best feed is given to abalone. Next, the cultured Ulva species will be compared with closely related seashore Ulva species and with Ulva specimens from the main farming area in Hermanus to see if they are genetically similar. Ulva specimens from these farms, nearby seashores, including the Hermanus abalone farming complex in the New Harbour were identified using morpho-anatomical and molecular methods. The molecular markers employed in this study were the plastid large subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL), Internal Transcribed Spacer of nuclear ribosomal DNA (ITS nrDNA) and the elongation factor tufA. The 12 cultured Ulva specimens belonged in the U. lacinulata clade with weak support value of 0.57 for PP in the rbcL tree, high support value of 0.86 for PP in the ITS tree and high support value of 92% and 0.92 for BP and PP, respectively, in the tufA tree. The seashore U. capensis and farmed Ulva specimens belonged in the same large U. lacinulata clade in the rbcL tree. However, the U. capensis samples and the locally cultivated Ulva samples belonged in separate sister clades with a support value of 70% and 0.75 for BP and PP, respectively, in the ITS phylogenetic tree, and 97% and 1 for BP and PP, respectively, in the tufA phylogenetic tree. Therefore, the identity of the cultivated Ulva samples is U. lacinulata and the clade containing the U. capensis samples has now been labelled as U. uncialis as it is an older available name than U. capensis. Furthermore, the foliose U. lacinulata was also found growing attached near the inlets of the Hermanus abalone farming complex in New Harbour. There was no genetic variation within the farmed Ulva samples as they were collapsed as a single haplotype by the three molecular markers. The genetic distance between the U. uncialis and farmed U. lacinulata samples were 0.16%, 0.76% and 0.92% for the markers, rbcL, ITS and tufA, respectively. Even though the low sequence divergence between the farmed U. lacinulata and U. uncialis specimens fits within the range of variability, these two clades are separate species that are closely related. Incongruences between the molecular and morpho-anatomical identification methods were observed, as the morpho-anatomical identification method identified 9 of the 12 farmed Ulva specimens as U. lactuca and the remaining as U. rigida sensu Stegenga et al. (1997). Fewer Ulva species were resolved morphologically because of the overlap in morphological description within U. lactuca sensu Stegenga et al. (1997) and U. rigida sensu Stegenga et al. (1997). Additionally, three new records of Ulva species (U. ohnoi, U. australis and U. stenophylloides) for South African seashore specimens were molecularly identified in this study, and foliose U. compressa was recorded for the first time in the region. In this study, the molecular marker tufA, was the best marker to delimit species, as its internal clades were better supported compared to the other two markers and it was able to better separate the farmed U. lacinulata samples and the seashore U. uncialis samples into two different clades.

Macroalgal blooms are increasing world-wide and have negative effects on benthic invertebrates and sediments. These include loss of species diversity and development of hostile sediment environments. This thesis considers ecological effects of Ulva lactuca L., and its mechanical removal on benthic invertebrates and sediments in Avon-Heathcote Estuary, New Zealand. Benthic communities comprised 34 species from 12 groups recorded from seven sites during seasonal general surveys. Dominant groups at each site were Gastropoda and Bivalvia. The most abundant species were Austrovenus stutchburyi, Micrelenchus tenebrosus and Amphibola crenata. Community composition varied significantly between sites, and there were significant site-specific differences in abundances of most species between winter and summer. U. lactuca had the greatest seasonal variation. Several species correlated with U. lactuca biomass, and the strength of correlation for different species varied between sites. There were seasonal changes in sediment physico-chemical variables between sites with greatest change in the silt/clay fraction. The sediment variables silt/clay fraction, dissolved oxygen and temperature correlated with seasonal changes of patterns in benthic community assemblages. A similar study was carried out by Bressington in 2003. In both studies, Bivalvia and Gastropoda were the most abundant groups, with Gastropoda having a higher, and Bivalvia a lower, proportion in the present study compared with 2003. Summer communities were significantly different between the two studies. Compared with 2003 there were higher percentages of sediment pore water and volatile solids present in 2005. Experimental removal of U. lactuca was conducted by mechanical broom at two sites: an open, exposed central sand flat, and McCormacks Bay, a shallow, sheltered mud flat. Removing U. lactuca had several immediate effects. These included a significant decrease in abundance of mobile epifauna (Micrelenchus tenebrosus and Zeacumantus subcarinatus) and an increase in abundance of infauna, including Arthritica bifurca and Austrovenus stutchburyi. There was no effect of U. lactuca removal on Austrovenus stutchburyi condition and 46 days following removal, invertebrate abundances approached pre-removal levels at each site. U. lactuca removal also caused short-term increases in dissolved oxygen and temperature of pore water. The greatest visual impact of removing U. lactuca was to sediments in McCormacks Bay from trampling. It was concluded that the variables having the greatest effect on seasonal species distribution and abundance at each site were temperature and sediment grain size. Differences between the present study and the study in 2003 were due to differences in sampling procedure mainly due to the two different quadrat sizes. Greater accuracy in representing long-term changes in ecosystems would be achieved by using standard sampling protocols. Removal of U. lactuca by mechanical broom was effective and had low impact on benthic invertebrates and physico-chemical variables, but it should be used only in sandy habitats because of severe disturbance to soft-sediment environments. Options for management and control of U. lactuca in Avon-Heathcote Estuary are discussed.

This study has investigated the gene expression of ubiquitin¡B20S proteasome beta subunit type 1 (20s£]1)¡Bubiquitin-conjugating enzyme e2 (ucee2)¡BATP-dependent caseinolytic protease regulatory subunit (clpC) in the marine macroalga Ulva fasciata Delile in relation to the hypersalinity-induced oxidative stress and protein oxidation. During the early stage (0-1 h), the water contents and TTC (2,3,5-tripheny tetrazolium chloride) reduction ability maintained unchanged but recovery ability and photosynthetic ability (PS II activity as indicated by Fv/Fm) were decreased along with accumulated H2O2, suggesting the occurrence of oxidative stress. Only ubiquitin expressed at this stage. During 1-3 h, water lost (approximately 33% of the control) with a further decrease in recovery ability, TTC reduction ability¡BPS II activity but more H2O2 accumulation and protein carbonyl compound. The transcripts of 20s£]1 and clpC and caseinolytic protease activity increased at this stage with the maximum of clpC at hour 3. In the 6-48 h, water lost seriously with high accumulated free amino acid at 6-12 h but low recovery ability. The transcript amounts of ubiquitin¡B20s£]1 and ucee2 increased marked during this stage, in which these might be related to programmed cell death caused by long-term exposure to hypersalinity. Reactive oxygen species (ROS) scavengers inhibited H2O2 accumulation, caseinolytic proteolytic activity increase, carbonyl compound formation and gene expression of ubiquitin¡B20s£]1¡Bucee2¡BclpC, indicating a role of ROS in the regulation of protease genes. A role of polyamines in the regulation of protease gene expression was tested. Spermidine and spermine inhibited the gene expression of ubiquitin¡B20s£]1 and ucee2, the oxidation of proteins (carbonyl groups) and the induction of caseinolytic protease activity in 90‰-treated thalli, whereas putrescine inhibited clpC expression, the oxidation of proteins and caseinolytic protease activity but enhanced the gene expression of ubiquitin¡B20s£]1 and ucee2. In conclusion, the results of the present investigation show that the degradation of oxidatively damaged proteins under hypersalinity conditions by increased caseinolytic protease activity is driven by the up-regulation of clpC gene expression via ROS and polyamines. It seems likely that the induction of ubiquitin¡B20s£]1 and ucee2 gene expression might be associated with the hypersalinity-mediated programmed cell death.

The effects of copper on growth and phosphorus (P) utilization were investigated in the marine chlorophyte Ulva fasciata Delile. Both the daily specific growth rate and tissue P contents decreased as increasing CuSO4 concentrations, while the contents of total tissue, intracellular and cell-wall Cu increased. Based on the relationship between daily specific growth rate and external CuSO4 concentrations, the upper limit of U. fasciata is 100 uM CuSO4. After 4-day exposure to varying CuSO4 concentrations, Pi uptake was inhibited. Analysis of P fraction in U. fasciata exposed to 100 uM CuSO4 shows that the Cu-induced decline in total tissue P contents is mainly due to a decrease in both soluble reactive P (i.e. Pi). Exposure to 100 uM of Cu caused the accumulation of total tissue Cu contents to a plateau and then rose again at day 3, and tissue P contents and daily specific growth rate decreased at day 4. IC50 (concentration of 50 % inhibition) of daily specific growth rate and tissue P contents are 9.8 and 37.8 uM of Cu concentration in the medium, respectively, and 0.76 and 1.44 mg¡Dg-1 DW for total tissue Cu contents, respectively. Overall, Cu causes Cu accumulation in intracellular space and cell wall and decrease of growth and P contents of U. fasciata partly via Pi uptake inhibition.

The roles of phosphorus (P) starvation on the induction of intracellular acid phosphatase (ACP; EC 3.1.3.2) activity have been studied in a marine macroalga Ulva lactuca L. In comparison to creasy and dark green appearance in P-sufficient thalli (100 mM NaH2PO4), P-starved thalli (1 mM NaH2PO4) showed less crease and light green appearance. On exposure to 1 mM NaH2PO4, the growth rate, the contents of SRP, PP and P, and tissue C:P and N:P molar ratio decreased at day 3 and the contents of SNRP, TSP and polyphosphate decreased immediately. Intracellular ACP activity increased at day 3 after exposure to 1 mM NaH2PO4 and reached 16 folds of P-sufficient thalli at day 14, while extracellular alkaline phosphatase (AP; EC 3.1.3.1) activity increased at day 2 and reached the plateau after 4 days. Activity staining both on Native PAGE and IEF gel showed the induction of 10 and 9 ACP isoenzymes, respectively. Changes in intracellular ACP and extracellular AP activities were negatively correlated with SRP, SNRP, PP and P contents. After transferred to 100 mM NaH2PO4, the growth rate of 10 day-starvated thalli recovered after 5 days, the contents of SRP¡BSNRP¡BTSP and P, and the C:P and N:P molar ratio recovered to the level of P-sufficient thalli at day 1. When recovered to 100 mM NaH2PO4, extracellular AP activity of 10 day-starvated thalli decreased at day 2 and reached the minimum after day 8, while intracellular ACP activity decreased at day 3 and reached the minimum after day 8. The analog of Pi, Phi (1 mM) inhibited the intracellular ACP and extracellular AP activities induced by P swtarvation. The results of present investigation show that ACP has a role in the enhancement of P availability in U. lactuca via the enzymatically degradation of polyphosphates and organic P when suffers P deficiency.

Macroalgal blooms are increasing world-wide and have negative effects on benthic invertebrates and sediments. These include loss of species diversity and development of hostile sediment environments. This thesis considers ecological effects of Ulva lactuca L., and its mechanical removal on benthic invertebrates and sediments in Avon-Heathcote Estuary, New Zealand. Benthic communities comprised 34 species from 12 groups recorded from seven sites during seasonal general surveys. Dominant groups at each site were Gastropoda and Bivalvia. The most abundant species were Austrovenus stutchburyi, Micrelenchus tenebrosus and Amphibola crenata. Community composition varied significantly between sites, and there were significant site-specific differences in abundances of most species between winter and summer. U. lactuca had the greatest seasonal variation. Several species correlated with U. lactuca biomass, and the strength of correlation for different species varied between sites. There were seasonal changes in sediment physico-chemical variables between sites with greatest change in the silt/clay fraction. The sediment variables silt/clay fraction, dissolved oxygen and temperature correlated with seasonal changes of patterns in benthic community assemblages. A similar study was carried out by Bressington in 2003. In both studies, Bivalvia and Gastropoda were the most abundant groups, with Gastropoda having a higher, and Bivalvia a lower, proportion in the present study compared with 2003. Summer communities were significantly different between the two studies. Compared with 2003 there were higher percentages of sediment pore water and volatile solids present in 2005. Experimental removal of U. lactuca was conducted by mechanical broom at two sites: an open, exposed central sand flat, and McCormacks Bay, a shallow, sheltered mud flat. Removing U. lactuca had several immediate effects. These included a significant decrease in abundance of mobile epifauna (Micrelenchus tenebrosus and Zeacumantus subcarinatus) and an increase in abundance of infauna, including Arthritica bifurca and Austrovenus stutchburyi. There was no effect of U. lactuca removal on Austrovenus stutchburyi condition and 46 days following removal, invertebrate abundances approached pre-removal levels at each site. U. lactuca removal also caused short-term increases in dissolved oxygen and temperature of pore water. The greatest visual impact of removing U. lactuca was to sediments in McCormacks Bay from trampling. It was concluded that the variables having the greatest effect on seasonal species distribution and abundance at each site were temperature and sediment grain size. Differences between the present study and the study in 2003 were due to differences in sampling procedure mainly due to the two different quadrat sizes. Greater accuracy in representing long-term changes in ecosystems would be achieved by using standard sampling protocols. Removal of U. lactuca by mechanical broom was effective and had low impact on benthic invertebrates and physico-chemical variables, but it should be used only in sandy habitats because of severe disturbance to soft-sediment environments. Options for management and control of U. lactuca in Avon-Heathcote Estuary are discussed.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 116-138). Also available via the World Wide Web.

Ingeniero Civil en Biotecnología / Hoy en día el tema de abastecimiento energético es cada vez más importante. En muchos países la matriz energética se basa en la explotación del recurso fósil, principalmente petróleo, el cual es un recurso limitado y que ha tenido una tendencia general al alza de precios. A lo anterior se añade las actuales normativas de emisiones que se están aplicando, las cuales prevén un futuro donde los combustibles fósiles se verán más limitados en su producción y aplicación. Los biocombustibles por tanto han sido una importante alternativa que se ha desarrollado por años, aunque, en un principio, muchos de los biocombustibles diseñados así competían (y compiten) en su materia prima con los recursos alimenticios (biocombustibles de primera generación). Además, una alternativa es el diseño de combustibles basado en residuos industriales, es decir materias primas que no son fuentes alimenticias (biocombustibles de segunda generación). Este estudio busca otra opción de producción de etanol, esta vez a partir de algas para producir biocombustibles de tercera generación, en especial se trabajó con la macroalga verde Ulva rigida. Para alcanzar el objetivo planteado, fue necesario diseñar un procedimiento experimental, el cual consistió en un pretratamiento con ácido diluido (1% y 2% v/v de ácido sulfúrico) a diferentes temperaturas (30°C y 120°C). Después del pretratamiento es necesario neutralizar las muestras para detener dicha reacción. La fracción sólida resultante del pretratamiento es sometida a una hidrólisis enzimática, gracias a la acción de celulasas y celobiasas durante 72 horas (Celluclast® 1,5L - Cat C2730, y Novozyme® 188 - CatC6105). Luego se fermentaron los azúcares liberados en la sacarificación mediante dos estrategias diferentes: Sacarificación y Fermentación Simultáneas (SSF), y Sacarificación Y Fermentación Separadas (SHF). Los resultados mostraron que el pretratamiento no es necesario; las muestras de algas utilizadas presentan azúcares fácilmente hidrolizables, y poseen una baja concentración de lignina, por lo que un pretratamiento con ácido diluido y temperatura no entrega mayor ventaja al proceso. Más aún, dicho procesamiento produce efectos negativos en la concentración de glucosa final. Por lo tanto, este proceso se suprimió del diseño planificado. La sacarificación de las algas sin pretratamiento presentó un rendimiento del 27,2% respecto a la glucosa total de la muestra. Por otra parte, los resultados de las dos estrategias de fermentación SSF y SHF fueron diferentes. El rendimiento, respecto a la conversión de glucosa total de la biomasa, de la estrategia SSF fue de un 50%, considerado alto para ser un primer acercamiento a la utilización de macroalgas. La estrategia SHF presentó un rendimiento del 24,5%. La diferencia de rendimientos indica que la estrategia SSF es mejor, aprovechándose de mejor forma los azúcares constituyentes de las macroalgas y obteniendo una mayor concentración de etanol en solución (5 g/l). Es importante mencionar que dicho rendimiento tiene su base en la composición total de glucosa de la macroalga, por lo que su alto valor puede corresponder a la fermentación de glucosa más la fermentación de otros azúcares no considerados en la composición total de glucosa. Aún así, la estrategia SSF es mejor que la SHF, ya que produce mayor etanol por gramo de biomasa empleado. A partir de los resultados obtenidos, se concluye que se logró un primer acercamiento a la producción de etanol a partir de la macroalga Ulva rigida, diseñándose un proceso de operación y evaluando el pretratamiento de ácido diluido en dicho procedimiento. También se concluyó que la estrategia SSF es mejor que la estrategia SHF para la producción de etanol a partir de la mencionada macroalga. Finalmente, se recomienda complementar este estudio mediante la caracterización completa de la macroalga utilizada.

As macroalgas desempenham importante papel no ambiente marinho como produtores primários. Elas ocupam diversas zonas do costão rochoso, entre elas, o mediolitoral que é a mais estudada devido às rápidas mudanças nos fatores ambientais (temperatura, dessecação, irradiância, salinidade, entre outros) direcionados pela dinâmica das marés. Esse dinamismo, fez com que os organismos desenvolvessem defesas que garantissem a sua sobrevivência nesses ambientes. E essas defesas, na maioria das vezes química, também possuem utilidade e aplicações biotecnológicas, pois as algas são fonte de novas substâncias e bioatividade com importância nutricional, médica ou farmacêutica. Como forma de reconhecer o potencial químico e bioativo ainda subexplorado nas algas brasileiras, o presente trabalho avaliou o potencial, antioxidante, antiviral, citotóxico, aspectos da fisiologia e a caracterização de ácidos graxos de três espécies de macroalgas do nordeste brasileiro: Sargassum vulgare (Ochrophyta), Palisada flagellifera (Rhodophyta), e Ulva fasciata (Chlorophyta) e seus potenciais foram testados para diferentes extratos. As espécies apresentaram bom potencial inibitório da enzima transcriptase reversa do vírus HIV e essa bioatividade se mostrou mais promissora, pois os extratos apresentaram baixa citotoxicidade frente ao crustáceo Artemia salina. O potencial antioxidante foi considerado bom para todas as espécies tanto para os extratos com finalidade química, tanto para os extratos com finalidade fisiológica, e este último, aliado às repostas fisiológicas de constituição pigmentar e proteínas sugerem que as algas desse estudo possuem uma boa aclimatação ao ambiente, pois não foi notada expressivas variações no seu conteúdo pigmentar. Quanto à caracterização de ácidos graxos, P. fagellifera se mostrou a maior fonte de ácidos graxos poliinsaturados (AGPIs) entre as espécies estudadas, o que faz dela um potencial recurso para obtenção de AGPIs com importância na suplementação alimentar como possíveis nutracêuticos e de importância médica / The macroalgae plays an important role in the marine environment as primary producers. They occupy different parts of the rocky shore, among them the midlittoral which is the most studied due to rapid changes in environmental factors (temperature, desiccation, irradiance, salinity, etc.) influenced by the dynamics of the tides. This dynamism, made the organisms developed defenses that guarantee their survival in these environments. These defenses, mostly chemical, also have utility and biotechnological applications because the algae are a source of new substances and bioactivity with nutritional importance, medical or pharmaceutical. As a way to recognize the chemical and bioactive potential still underexploited in Brazilian seaweed, this study evaluated the potential, antioxidant, antiviral, cytotoxic, aspects of physiology and fatty acids characterization of seaweeds in northeastern Brazil Sargassum vulgare (Ochrophyta) Palisada flagellifera (Rhodophyta) and Ulva fasciata (Chlorophyta) tested for different extracts. The species showed good potential as inhibitors of the enzyme reverse transcriptase of HIV and this bioactivity was more promising because the extracts showed low cytotoxicity against the crustacean Artemia salina. The antioxidant potential was considered good for all species for both the extracts with chemical purposes, for both the extracts with physiological purpose, and the latter coupled to the physiological responses of pigment composition and proteins suggest that the algae of this study have good acclimation environment, it was noted significant variations in their pigment content. The characterization of fatty acids, P. fagellifera showed the largest source of polyunsaturated fatty acids (PUFAs) among the studied species, making it a potential resource for obtaining PUFAs of importance in the food supplementation as possible nutraceutical and medical importance