Cyanobacteria are classified as alkalophiles despite their preferential uptake of the acidic form of dissolved inorganic carbon. Long term impacts of external pH on the expression of photosynthetic and structural pigments in Schizothrix calcicola were investigated as potential contributing factors to this phenomenon. More robust cell walls in S. calcicola at pH <7 are suggested by significantly greater expression of myxoxanthophylls. Direct and indirect physiological costs of altering cell walls may contribute to S. calcicola's depressed growth at acidic pH. Comparison of chlorophylls expression suggests that alkaline rather than neutral external pH is only beneficial for S. calcicola growth in absence of nutrient limitation. While the cyanobacterial biomarker ratio of chlorophylls to echinenone was stable across the pertinent pH range of 6-8, other pigment ratios in S. calcicola were affected by pH with an approximately two week lag between the change of pH and the corresponding change of pigment expression. / by Maria West. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Gas fermentation has emerged as a promising technology that converts waste gases containing CO, CO2 and H2 (also known as syngas) into fuels and chemical commodities. Employed by LanzaTech Inc., Clostridium autoethanogenum is an industrial acetogen that converts gases into ethanol, 2,3-butanediol, acetate, and lactate. Metabolic engineering offers unique opportunities to eliminate side-products, synthesize novel, high-value molecules as diversification strategies, and increase productivities of natural products. However, there had been no scientific reports of genetic manipulation of this acetogen so the overall goal of this PhD project was to develop genetic tools for this gas-utilizing microorganism and construct a hyper-ethanol producing strain via metabolic engineering. The formulation of electroporation and conjugation procedures allowed exogenous DNA to be routinely introduced into the bacterial host. ClosTron mutagenesis and Allele-Coupled Exchange (ACE) techniques were fully exemplified in this bacterium during the construction of knockout, in-frame deletion, and overexpression mutants. Carbon monoxide dehydrogenases (cooS1, cooS2 and acsA) were specifically targeted to elucidate their roles in supporting CO oxidation and carbon fixation. In the ethanol formation pathway, inactivation of bi-functional aldehyde/alcohol dehydrogenases (adhE1 and adhE2) impaired growth on pure CO but elevated ethanol titres. Conversely, inactivation of the more highly expressed aldehyde:ferredoxin oxidoreductase (aor1), but not the weakly expressed aor2, significantly reduced ethanol production, highlighting the importance of aor1 in autotrophic ethanol formation. A double KO mutant of aor1 and aor2 was also generated via ClosTron mutagenesis and pyrE-mediated allelic exchange. In an effort to engineer a robust biocatalyst, the native chaperone systems groESL and/or grpE-dnaK-dnaJ were overexpressed in C. autoethanogenum, resulting in enhanced tolerance towards ethanol, heat and salts. In summary, this study demonstrated the genetic tractability of C. autoethanogenum and revealed gene targets for future metabolic engineering of a hyper-ethanol producing acetogen.

A intensificaÃÃo da eutrofizaÃÃo nos mananciais à provocada pelas aÃÃes antrÃpicas e tem como predominÃncia as floraÃÃes de cianobactÃrias. Por sua vez, estas floraÃÃes alteram a qualidade da Ãgua para o abastecimento da populaÃÃo, e, ao serem lisadas, podem liberar toxinas (cianotoxinas) causando intoxicaÃÃo. A primeira confirmaÃÃo in loco da morte de seres humanos por intoxicaÃÃo com cianotoxinas no Brasil levou à OMS a publicar rapidamente novas Portarias sobre o monitoramento da qualidade da Ãgua bruta que incorporaram novos indicadores, como a concentraÃÃo das cianobactÃrias e de suas toxinas nos mananciais utilizados para abastecimento de Ãgua potÃvel. Com isso, as tecnologias para o tratamento da Ãgua e para a identificaÃÃo e quantificaÃÃo das cianotoxinas vÃm passando por um processo de aperfeiÃoamento. Neste contexto, o presente trabalho objetivou validar o mÃtodo de cromatografia lÃquida de fase reversa com detector de fluorescÃncia (CLAE-FLD) e derivatizaÃÃo prÃ-coluna para detecÃÃo e quantificaÃÃo das cianotoxinas saxitoxina (STX) e decarbamoil-saxitoxina (dc-STX) proveniente do cultivo da cianobactÃria Cylindrospermopsis raciborskii. Esta validaÃÃo foi realizada para dar credibilidade ao mÃtodo analÃtico e os parÃmetros selecionados foram: seletividade, linearidade, limite de detecÃÃo (LD) e quantificaÃÃo (LQ), exatidÃo, precisÃo e robustez. Os resultados obtidos apresentaram boa seletividade, comprovando que o mÃtodo possuÃa capacidade de medir as toxinas em uma matriz PÃs ExtraÃÃo na presenÃa de outros componentes. As curvas analÃticas foram construÃdas com nove pontos a partir dos padrÃes de STX e dc-STX. O mÃtodo apresentou uma linearidade no intervalo de 4,5 à 150 Âg L-1 para STX e 3,0 à 132 Âg L-1 para dc-STX, e o coeficiente de correlaÃÃo (r) maior que 0,99 para as duas toxinas, mostrando que o mÃtodo tem a capacidade de fornecer resultados diretamente proporcionais à concentraÃÃo dos analitos detectados. A sensibilidade foi medida atravÃs do LD e LQ, obtendo resultados satisfatÃrios para os objetivos do trabalho. O mÃtodo obteve boa precisÃo e exatidÃo, visto que para STX e dc-STX os diferentes nÃveis de concentraÃÃo estavam com valores dentro dos intervalos permitidos pelas normas brasileiras de validaÃÃo, e apresentou-se robusto, pois foi insensÃvel a pequenas variaÃÃes possÃveis de ocorrer durante a anÃlise. Em resumo, pode-se considerar que o mÃtodo utilizado para a detecÃÃo e quantificaÃÃo das cianotoxinas STX e dc-STX apresentou resultados satisfatÃrios, uma vez que os parÃmetros analisados para validÃ-lo estavam em conformidade aos valores aceitos nas normas brasileiras. / The intensification of eutrophication in the watershed is caused by human actions and is the predominant cyanobacteria. In turn, these blooms affecting the quality of the water supply for the population, and, when disrupted, can release toxins (cyanotoxins) causing intoxication. The first in situ confirmation of the death by poisoning of humans with cyanotoxins in Brazil led the WHO to quickly publish new Ordinance on monitoring of raw water quality that incorporate new indicators, as the concentration of the cyanobacteria and their toxins in water sources used for drinking water supply. Thus, technologies for water treatment and for the identification and quantification of cyanotoxins have been going through a process of improvement. In this context, this study aimed to validate the method of reverse phase liquid chromatography with fluorescence detection (HPLC-FLD) and pre-column derivatization for detection and quantification of cyanotoxins saxitoxin (STX) and decarbamoil-saxitoxin (dc-STX) from the cyanobacterium Cylindrospermopsis raciborskii cultivation. This validation was performed to give credibility to the analytical method and the selected parameters were: selectivity, linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision and robustness. The results showed good selectivity, confirming that the method had the ability to measure the toxins in a Post Extraction matrix in the presence of other components. The analytical curves were constructed with nine points from the patterns of STX and dc-STX. The method showed linearity in the range of 4.5 to 150 mg L-1 for STX and 3.0 to 132 mg L-1 to dc-STX, and the correlation coefficient (r) greater than 0.99 for both toxins, showing that the method has the capacity to deliver results directly proportional to the concentration of analyte detected. The sensitivity was measured by the LD and LQ, obtaining satisfactory for the purposes of work results. The method achieved good precision and accuracy, whereas for STX and dc-STX different concentration levels were with values &#8203;&#8203;within the ranges allowed by Brazilian standards for validation, and showed to be robust because it was insensitive to small variations possible to occur during analysis. In summary, one can consider that the method used for the detection and quantification of cyanotoxins STX and dc-STX showed satisfactory results, since the parameters analyzed to validate it were in conformity with the accepted values &#8203;&#8203;in Brazilian standards.

O objetivo deste estudo foi verificar se existe associação entre a qualidade da água para abastecimento público proveniente do Sistema Guarapiranga e o clima da Região Metropolitana de São Paulo (RMSP). Realizou-se a análise dos dados meteorológicos adquiridos junto à Estação Meteorológica do Instituto de Astronomia, Geofísica e Ciências Atmosféricas da Universidade de São Paulo (IAG/USP) e dos resultados de análises laboratoriais da água do Reservatório Guarapiranga adquiridos junto à Companhia de Saneamento Básico do Estado de São Paulo (SABESP). A partir da relação entre as variáveis observadas em gráficos e testes de associação/correlação, verificou-se a associação/correlação entre a densidade de cianobactérias e as variáveis meteorológicas na RMSP, tais como temperatura atmosférica (T°C), insolação (horas de brilho do sol) e precipitação (mm). Para analisar as interações e relações de cada uma das variáveis meteorológicas em relação à densidade de cianobactérias, foram feitos diferentes recortes de tempo. Todos os dados foram organizados em Planilhas Microsoft® Excel 15.0 (Office 2013) e analisados em gráficos e testes estatísticos. Segundo os resultados deste estudo, a densidade de cianobactérias apresenta associação positiva com os períodos de chuva e temperaturas elevadas (outubro a março) e, juntamente com as análises de dados climáticos dos últimos 42 anos, verificou-se que as condições climáticas ideais para a proliferação de cianobactérias no Reservatório Guarapiranga vêm se acentuando ao longo das últimas quatro décadas, particularmente nos últimos 20 anos. Conclui-se que há indicações de que a densidade de cianobactérias no Reservatório do Sistema Guarapiranga tenha relação temperaturas mais elevadas e pluviosidade e que a proliferação de cianobactérias pode aumentar caso se mantenha a tendência do clima. / This study has aimed to verify if there is a relation between the quality of the water in the Guarapiranga System (reservoir), as it is supplied to the inhabitants, and the climate in the São Paulo Metropolitan Area (RMSP). We have analyzed the data obtained from the Weather station of the Astronomy, Geophysics and Atmospherical Sciences Institute at the University of São Paulo (IAG/USP), as well as the results of the reservoir water laboratory analysis, provided by the basic sanitation company in the State of São Paulo (SABESP). Cyanobacteria density and meteorological variables in the RMSP, such as atmospheric temperature (ToC), insolation (daily solar irradiance) and precipitation (mm), were verified through association/correlation tests and graphics. To analyze the interaction and relation between cyanobacteria density and meteorological variables, were have resorted to different time spans. Data was organized in Microsoft® Excel 15.0 (Office 2013) tables and graphics, and statistically analyzed. According to this study results, cyanobacteria density might be positively associated to periods of rainfall and high temperatures (October to March). Also, along with data analysis of climate throughout the last 42 years, we have observed that the ideal climate conditions for cyanobacteria proliferation in the Guarapiranga reservoir have been stressed in the last four decades, especially during the last 20 years. Therefore, frequency and intensity of cyanobacteria proliferation in the Guarapiranga reservoir may increase according to climate trend in the RMSP.

Orientador: Maria do Carmo Bittencourt de Oliveira / Banca: Orlando Necchi Junior / Banca: Daniel Scherer de Moura / Resumo: Florações de cianobactérias tóxicas são atualmente um problema mundial devido a produção de toxinas. A microcistina é uma hepatotoxina comumente encontrada em corpos d'água e é produzida principalmente pelo gênero Microcystis. Recentemente a identificação, clonagem e seqüenciamento do agrupamento de genes responsáveis pela codificação da sintetase de microcistina (MS) tornaram possível a abordagem molecular na detecção de populações tóxicas baseada na técnica de PCR (Polymerase Chain Reaction). A técnica de PCR tornou-se viável e útil pelo fato do agrupamento de genes da sintetase de microcistina estar presente apenas em organismos tóxicos e pela ausência de diferenças morfológicas entre aqueles tóxicos e não tóxicos. Este estudo objetivou desenvolver e avaliar a técnica de PCR competitiva para quantificação de células de Microcystis tóxicas e não tóxicas utilizando os genes cpcBA e mcyB envolvidos respectivamente, na formação da ficocianina e biossíntese da MS. Testou-se a hipótese de que a PCR competitiva pode ser utilizada como metodologia de quantificação de células tóxicas e não tóxicas de Microcystis em substituição a contagem direta de células por microscopia óptica para atender a Portaria do Ministério da Saúde 518/2004. Para obtenção de DNA competidores foram realizadas amplificações seqüenciais de "células DNA equivalente" das linhagens tóxica (BCCUSP18) e não tóxica (BCCUSP03) de Microcystis spp. utilizando primers descritos na literatura, bem como aqueles desenhados para este estudo. Avaliaram-se os DNA competidores amplificando-os com células DNA equivalente das linhagens. Após determinada a quantidade mais próxima de DNA competidores para quantificar 2,0 x 104 células, realizou-se a PCR competitiva com células DNA equivalente de uma amostra ambiental (reservatório de Duas Unas, PE)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Blooms of microcystin-producing cyanobacteria are a problem worldwide. Mycrocystin is a liver hepatotoxin commonly found in bodies of water and is produced mainly by the genus Microcystis. The recent identification, cloning and sequencing of the genes responsible for coding microcystin synthetase (MS) has allowed a molecular approach to the detection of micocystin-producing populations based on the Polymerase Chain Reaction (PCR) method. Considering the lack of morphological differences between microcystin-producing and non-microcystin-producing organisms, PCR is viable and useful, as the cluster of microcystin synthetase genes is found in only microcystinproducing organisms. The aim of the present study was to develop and assess an competitive PCR method for the quantification of toxic and non-toxic Microcystis cells using the cpcBA and mcyB genes, which are respectively involved in the formation of phycocyanin and biosynthesis of MS. The hypothesis was that competitive PCR could be used as a quantification method for toxic and non-toxic Microcystis cells, replacing the direct cell count under an optical microscope, while fulfilling Brazilian Ministry of Health Ordinance 518/2004. For the acquisition of competitor DNA, sequences amplifications were carried out of the "cell DNA equivalent" of microcystin-producing (BCCUSP18) and non-microcystin-producing (BCCUSP03) strains of Microcystis spp. using primers described in the literature as well as others designed for the present study. Competitor DNA was assessed by amplifying "cells DNA equivalent" of the strains. After determining the quantity closest to the competitor DNA for quantifying 2.0 x 104 cells, competitive PCR was carried out with "cells DNA equivalent" from an environmental sample (Duas Unas Reservoir, PE, Brazil). The constructed competitor cpcBA quantified 1.45 x 104 cells (R2= 0.989)... (Complete abstract click electronic access below) / Mestre

The stromatolites of Shark Bay Western Australia, located in a hypersaline environment, is an ideal biological system for studying survival strategies of cyanobacteria and halophilic archaea to high salt and their metabolic cooperation with other bacteria. To-date, little is known of the mechanisms by which these stromatolite microorganisms adapt to hypersalinity. To understand the formation of these sedimentary structures, detailed analysis of the microbial communities and their physiology for adaptation in this environment are crucial. In this study, microbial communities were investigated using culturing and molecular methods. Phylogenetic analysis of the 16S rRNA gene was carried out to investigate the diversity of microorganisms present. Unique phylotypes from the bacteria, cyanobacteria and archaea clone libraries were identified. Representative cyanobacteria isolates and Halococcus hamelinensis, a halophilic archaea isolated from in this study, were the focus for identifying osmoadaptation mechanisms. The presence of osmolytes in these microorganisms was detected by Nuclear magnetic resonance spectroscopy (NMR). It was found that the cyanobacterial isolates studied utilised different osmolytes. Glucosylglycerol, unique to marine cyanobacteria was not identified; instead various saccharides, glycine betaine and TMAO were the predominant solutes present. Thus cyanobacteria are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. Findings here also indicated that H. hamelinensis accumulates glycine betaine and glutamate instead of potassium ions. DNA molecular methods were employed to identify candidate genes for the uptake of osmoprotectants. Three putative glycine betaine transporters from Halococcus hamelinensis were identified. Functionality of one of these glycine betaine transporters was determined by complementation studies. For the first time, an archaeal glycine betaine transporter was shown to be successfully complemented in a glycine betaine transport deficient mutant (E. coli MKH13). This study has increased our understanding of how microorganisms co-exist in fluctuating environments in response to solubilisation/precipitation or dilution/evaporation processes, resulting in a hypersaline environment. It also provides an excellent platform for the identification of any novel osmolytes/compatible solutes that might have been produced by these microorganisms that have been isolated for the first time from stromatolites.

Graduation date: 2001

Graduation date: 1998

Two decades ago the recruitment of YOY perch (Perca fluviatilis) started to decline along the Swedish east cost of the Baltic Sea. Factors that influence recruitment are e.g. eutrophication that causes habitat losses and overfishing of cod (Gadus morhua) which causes cascading effects in the food web. Filamentous cyanobacterial blooms are often toxic and has increased in the Baltic Sea and its coastal waters. The aim of this field study was to evaluate the effects of salinity and cyanobacterial exposure on fitness related parameters of young of the year (YOY) perch (Perca Fluviatilis) in a natural environment. Our study was performed in the Curonian Lagoon (Lithuania) in August 2009. The lagoon offers a temporary salinity gradient (wind induced influxes from the Baltic Sea) ranging from 7 psu in the north to 0 psu in the south. Submerged enclosures containing YOY perch were set up at three different locations along the salinity gradient in the Lagoon (referred to as North, Middle, South). The duration of the experiment was 21 or 27 days, depending on treatment. Measurements of perch condition were specific growth rate, somatic condition index (SCI) and whole fish lipid and protein content. Average chl a values for the three stations during the experimental time were: north 180 ± 70 µg/l chl a, middle 133 ± 36 µg/l chl a and south 180 ± 52 µg/l chl a. The North and the Middle stations experienced two different salinity influxes reaching a maximum salinity of 6.5 psu at the northern station. The duration of each saline influx was approximately 4-6 days. The saline water did not reach the Southern station at any time. Results show that perch from the southern station were in best condition in terms of specific growth rate and contents of total lipids. Compared to the South the perch condition declined to the Middle station and was lowest at the Northern station which experienced the highest degree of fluctuation in terms of salinity and cyanobacterial exposure. Examination of the abundance of the main food resource at the different stations revealed no statistical differences, which suggest that availability of food was not a factor in explaining the differences in growth.  The results possibly indicate that a changing environment with the potential synergistic negative effects of salinity and cyanobacteria has a higher negative impact on YOY perch condition compared to constantly high concentrations of cyanobacteria.

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