This research investigated the seasonal and spatial distributions of picophytoplankton, including Prochlorococcus, Synechococcus and picoeukaryotes, in the northern South China Sea. Monitoring experiments including light intensity control and nutrient enrichment were conducted concurrently with on board sampling to examine factors affecting their cell densities dynamics. Quantification of cell numbers was carried out by flow cytometry. Averaged Synechococcus abundance in the South China Sea was 1¡Ñ104 cells ml-1, high in winter (1.37¡Ó0.30¡Ñ104 cells ml-1) and low in summer or fall (0.51¡Ó0.13¡Ñ104 cells ml-1 and 0.53¡Ó0.22¡Ñ104 cells ml-1, respectively). During a same season of the year, there was more Synechococcus in the shelf-slope region than in the basin. The cell density in summer, but not in winter, was significantly positively related to surface water nutrient concentration. Nutrient enrichment experiment carried out in winter also indicated that the growth of Synechococcus did not respond to addition of nitrate. On the other hand, Synechococcus seemed to prefer high illumination. In the light intensity experiment, Synechococcus collected from surface water grew better at 100% surface illumination than <100% light intensities. Synechococcus collected from deep water grew best at 30% and 18% of surface illuminations. Vertically, Synechococcus concentrated mostly in surface water with maximum cell number occurring at the surface or a few meters deep. Nutrient enrichment experiment in winter also showed that Synechococcus responded significantly to iron addition. Average cell density of picoeukaryotes was always less than 0.5¡Ñ104 cells ml-1, being high in winter (0.46¡Ó0.10¡Ñ104 cells ml-1) and low in summer or fall (0.15¡Ó0.02¡Ñ104 cells ml-1 and 0.19¡Ó0.03¡Ñ104 cells ml-1, respectively). Picoeukaryotes was always more concentrated in the shelf-slope region than in the basin, especially in winter when cell density in the shelf-slope region was 0.70¡Ó0.11¡Ñ104 cells ml-1. Although in winter picoeukaryotes was significantly positively related to surface water nutrient concentration, enrichments of nitrate or iron did not enhance their growth. Prochlorococcus had a cell density > 5.5¡Ñ104 cells ml-1 in the euphotic zone, and distributed as deep as 200 m. Light intensity monitoring experiment showed that Prochlorococcus from surface water grew better under high illumination than those from deep water and vice versa. Under 9% of surface illumination, deepwater Prochlorococcus population showed a positive growth, corresponding well with its deep distribution. Nutrient enrichment experiment conducted in winter showed that Prochlorococcus did not respond to enrichment of nitrate or iron.

Cyanobacteria are photosynthetic prokaryotes that live in aquatic environments. The cyanobacterium Synechococcus elongatus PCC 7942, (hereafter S. elongatus) coordinates its day and night behaviors via a circadian clock. The clock is entrained by light/dark cycles but continues to run in constant light conditions. The core circadian clock in S. elongatus is encoded by post-translational modifications of three Kai proteins, but the extent and mechanism of circadian gene expression are unknown. We provide the first unbiased characterization of circadian gene expression in S. elongatus, demonstrating that \(\sim 65\%\) of genes display oscillation in continuous light conditions, with some genes peaking in expression at subjective dawn and others at subjective dusk. We next sought to identify the mechanism by which such a large fraction of the genome could be rhythmically controlled. Through bioinformatic, correlative, and perturbation experiments, we find that circadian changes in chromosome topology/supercoiling are sufficient to drive rhythmic expression (Chapter 2). To further investigate how chromosome topology can control gene expression we performed a high resolution characterization of transcripts and RNA polymerase across the S. elongatus genome (Chapter 3). Bioinformatic analysis of transcription start sites suggests that the AT/GC content a particular region of the promoter is informative in defining the phase at which a transcript is maximally expressed. We find that these sequences are sufficient to drive circadian gene expression at a particular phase and that mutation of single nucleotides in this region can reverse the expression phase of a transcript (Chapter 4). To understand the role of chromosome dynamics in circadian gene expression and cyanobacterial physiology, we tagged and followed chromosomes over multiple cell divisions. We find that S. elongatus cells harbor multiple ordered copies of a single chromosome, and the organization of chromosomes in the cytoplasm facilitates equal segregation of chromosomes to daughter cells (Chapter 5).

Apesar do cultivo de camarÃo ser uma importante atividade econÃmica, o seu crescimento tem atraÃdo a atenÃÃo para os possÃveis impactos ao meio ambiente. As Ãguas residuais geradas pelos cultivos podem contribuir para a eutrofizaÃÃo das Ãguas receptoras desses efluentes, jà que possuem uma ampla variedade de substÃncias consideradas contaminantes potenciais, como o metabissulfito de sÃdio, um agente sulfitante que retira o oxigÃnio da Ãgua e de alimentos. AlÃm disso, os efluentes carreiam a comunidade fitoplanctÃnica que prolifera nos sistemas de cultivo, incluindo cianobactÃrias produtoras de toxinas. Com isso o objetivo desse estudo foi uma avaliaÃÃo ecotoxicolÃgica utilizando diferentes organismos testes e respostas biolÃgicas dos possÃveis impactos causados pelo uso do metabissulfito de sÃdio ou pela proliferaÃÃo de cianobactÃrias coletadas nos prÃprios cultivos. Amostras do fitoplÃncton foram coletadas em trÃs fazendas do estado do Cearà para anÃlise qualitativa, quantitativa e para o isolamento das cianobactÃrias. Estes organismos foram identificados atravÃs de tÃcnicas morfolÃgicas e de biologia molecular, cultivados no laboratÃrio para obtenÃÃo dos extratos e realizados ensaios com camundongos e misidÃceos. O metabissulfito de sÃdio teve sua toxicidade avaliada por meio de ensaios agudos em diferentes nÃveis trÃficos (microalgas, cladÃceros, camarÃes e peixes), mas tambÃm pela taxa do consumo de oxigÃnio e das atividades enzimÃticas (CAT; AChE e GST) em camarÃes. Para os peixes, foram realizados ainda ensaios com o desenvolvimento embriolarval e das atividades enzimÃticas (AChE e GST). A anÃlise qualitativa e quantitativa da comunidade fitoplanctÃnica mostrou um grande nÃmero de espÃcies de quase todas as classes do fitoplÃncton, inclusive cianobactÃrias. Deste Ãltimo grupo foi isolada e cultivada Synechococcus sp., para a qual o extrato e as cÃlulas vivas mostraram toxicidade em camundongos evidenciada por danos observados no fÃgado e rins dos animais. Jà na avaliaÃÃo da toxicidade do metabissulfito de sÃdio, este apresentou toxicidade aguda para os organismos testes, apresentando uma EC50 de 24,29 mg/L para as microalgas, CL50 de 34,90 mg/L e 69,65 mg/L para cladÃceros e camarÃes, respectivamente. Em relaÃÃo ao consumo de oxigÃnio e atividades enzimÃticas, estes nÃo apresentaram diferenÃas significativas quando comparados aos controles, com exceÃÃo para a AChE nos camarÃes e GST nos peixes. No desenvolvimento embriolarval dos peixes, o metabissulfito de sÃdio causou diferentes tipos de efeitos desde a destruiÃÃo dos ovos à deformidades nas larvas. Sendo assim, esses resultados mostram que o metabissulfito de sÃdio e a cianobactÃria Synechococcus sp. sÃo tÃxicos para diferentes organismos, evidenciados tanto pela toxicidade aguda quanto pela crÃnica. / Farming of shrimp is an important economic activity; however, its exponential growth has attracted attention to the potential impacts brought over the environment. Wastewater generated by this activity can contribute to the eutrophication of waters receiving these effluents, as those may bear a variety of contaminants, such as sodium metabisulfite, a sulphitant agent that removes the oxygen from water and food. Additionally, this effluent carries the phytoplankton community that proliferates in these systems, which may include toxic cyanobacteria. Therefore, the aim of the present study was to perform an ecotoxicological assessment using biological test models and biological responses to evaluate potential impacts caused by use of sodium metabisulfite and by the proliferation of cyanobacteria in shrimp farming. Phytoplankton samples were collected in three shrimp farms from the state of Cearà for quantitative and qualitative analysis and for the isolation of cyanobacteria. The microorganisms, which were identified by morphological and molecular techniques, were grown in laboratory to o btain organic extracts on which were conducted toxicity tests with mice and mysids. Sodium metabisulfite was evaluated through acutetoxicity tests using organisms at different trophic levels (microalgae, cladocerans, shrimps and fish) and by the rate of oxygen consumption and enzymatic activities (CAT,AChE and GST) in shrimps. On fish, tests evaluating the embriolarval development and enzymatic activities (AChE and GST) were performed. Qualitative and quantitative analysis of phytoplankton community showed a large number of species from nearly all classes of phytoplankton, including cyanobacteria. From this latter group, Synechococcus sp. was isolated and cultivated, and the extract generated from the living cells showed toxicity in mice, as evidenced by damage to the liver and kidneys of animals. For acute toxicity tests sodium metabisulphite showed an EC 50 of 24.29 mg/L for the microalgae and LC 50 of 34.90 mg/L and 69.65 mg/L for cladocer ans and shrimp, respectively. Regarding the consumption of oxygen and enzymatic activities, these did not show significant differences when compared to their respective controls, except for the AChE in shrimp and GST in fish. On embriolarval developing of fish, sodium metabisulfite caused different types of effects, from destruction of eggs to deformities in larvae. Thus, these results show that sodium metabisulphite and the cyanobacteria Synechococcus sp. are toxic to other organisms, evidenced by both, acute and chronic toxicity

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2007. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-25T21:54:08Z No. of bitstreams: 1 cultivo de microalgas com gases de combusto formados da gerao termeltrica.pdf: 1291931 bytes, checksum: 947e8e39923ea39e3dd24f11490dae18 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-11-07T00:58:29Z (GMT) No. of bitstreams: 1 cultivo de microalgas com gases de combusto formados da gerao termeltrica.pdf: 1291931 bytes, checksum: 947e8e39923ea39e3dd24f11490dae18 (MD5) / Made available in DSpace on 2012-11-07T00:58:29Z (GMT). No. of bitstreams: 1 cultivo de microalgas com gases de combusto formados da gerao termeltrica.pdf: 1291931 bytes, checksum: 947e8e39923ea39e3dd24f11490dae18 (MD5) Previous issue date: 2007 / O aumento da concentração de gás carbônico na atmosfera tem sensíveis conseqüências ambientais. Nos últimos anos a emissão de CO2 na atmosfera aumentou de 280ppm (1800) para 380ppm (2004), sendo cerca de 22% dessas emissões causadas por plantas de energia termelétrica. Dentre as várias alternativas para captura e utilização de CO2, uma abordagem particularmente interessante é o emprego de microalgas. As microalgas se destacam por apresentarem diversas potencialidades, como fonte de alimento e fonte para obtenção de bioprodutos, e também podem contribuir na redução do efeito estufa, fixando CO2. As microalgas Chlorella e Spirulina apresentam em sua composição alto teor de proteínas, ácidos graxos, sais minerais e pigmentos, e além disso, possuem certificado GRAS(Generally Recognized As Safe), podendo ser utilizadas como alimento sem oferecer risco à saúde humana. A captura do CO2 do gás de combustão de carvão é possível usando microalgas, tanto por separação como por uso direto do gás de combustão, sendo este último mais vantajoso, em função de uma maior economia de energia. Alguns agravantes podem influenciar no uso direto do gás de combustão como a alta temperatura, concentração de CO2 acima de 15% e a presença de SOx, NOx e material particulado (em especial cinzas), dificultando assim, o método direto, a menos que a microalga suporte condições extremas. O objetivo deste trabalho foi estudar a utilização de gases de combustão do carvão provenientes da geração termelétrica, para cultivo de microalgas. Previamente foi realizada seleção de microalgas quanto à resistência a SO2 que pode ser formado da combustão do carvão para geração de energia elétrica. As microalgas estudadas foram Chlorella homosphaera, Scenedesmus obliquus e Spirulina sp. expostas a de 6% de CO2 e 30ppm de SO2. A máxima produtividade de biomassa alcançada foi 0,19 g.L-1.d-1 e concentração celular máxima 2,92 g.L-1, ambos para microalga Spirulina sp. Após estudou-se as microalgas S. obliquus e Spirulina sp. em um sistema de FBRs em série em diferentes concentrações de CO2, SO2, NO e diferentes temperaturas. Foi alcançada concentração celular média máxima de 3,29 g.L-1 e fixação de CO2 máxima de 35,87%, ambos resultados para Spirulina sp. Seguindo o estudo em biofixação de CO2 por microalgas, foram isoladas as microalgas Synechococcus nidulans e Chlorella vulgaris da lagoa de estabilização da Usina Termelétrica Presidente Médici – UTPM/CGTEE, sul do Brasil. As microalgas isoladas foram cultivadas e comparadas com as microalgas Spirulina sp e S.s obliquus, em relação a biofixação de CO2. As microalgas foram expostas a 12% CO2, 60 ppm de SO2 e 100 ppm de NO, simulando um gás de combustão de carvão. A C. vulgaris apresentou comportamento semelhante a Spirulina sp., alcançando 13,43% de fixação diária máxima. Foi determinado o conteúdo lipídico e a composição em ácidos graxos das microalgas Spirulina sp., S. obliquus, S. nidulans e C. vulgaris cultivadas em meio contendo 12% de CO2, 60 ppm de SO2 e 100 ppm de NO à 30ºC. A microalga S. obliquus apresentou o maior teor lipídico (6,18%). Para as demais microalgas o conteúdo lipídico variou de 4,56 a 5,97%. O maior conteúdo em AGMI foi 66,01% para a S. obliquus. Os ácidos graxos poliinsaturados (PUFA) foram alcançados em maior quantidade pelas microalgas Spirulina sp. (29,37%) e S. nidulans (29,54%). Os resultados mostraram que o cultivo de microalgas enriquecido com os gases CO2, SO2 e NO, apresentaram uma biomassa rica em ácidos graxos, podendo estes ser utilizados tanto para a alimentação (ácidos graxos insaturados), quanto para produção de biocombustíveis(ácidos graxos saturados). Além disso, as microalgas estudadas podem contribuir na redução do aquecimento global. / The increasing concentration of carbon dioxide in the atmosphere has sensible environmental consequences. In the recent years the concentration of CO2 in the atmosphere increased from 280ppm (1800) to 380ppm (2004), around 22% of these emissions caused by coal fired power plants. Amongst several alternatives for the capture and application of the CO2, one of the most interesting overviews it is the use of microalgae. Microalgae are gain eminence for presenting potentiality, like a source of nutrients and for biofuels production, besides, they can contribute with the greenhouse gas abatement, fixing CO2. Chlorella and Spirulina presents a high amount of proteins, fatty acids, minerals and pigments in their composition, besides, they have the GRAS certificate (Generally Recognized As Safe), allowing them to be used like food without offer any risk to the human health. The CO2 capture from the coal fired flue gas is possible, as by the separation of the CO2 as by the direct use of the flue gas, being the last one advantageous, due to the major energy economy. Some bottlenecks can influence the direct use of the flue gas like the high temperature of the gas, high CO2 concentration and the presence of SOx, NOx and particulate matter(specially ashes), becoming hard, thus the direct method, unless that the microalga could tolerate extreme conditions. The aim of this work was to study the utilization of coal fired flue gas from power plants in microalgal cultures. Previously, was carried out the selection of the microalgae resistant to SO2, witch can be formed in the coal fired power generation. The studied microalgae were Chlorella homosphaera, Scenedesmus obliquus and Spirulina sp. LEB-18 exposed to 6% CO2 and 30ppm SO2. The maximum biomass productivity was 0.19 g.L-1.d-1 and the maximum cell concentration was 2,92 g.L-1, both for Spirulina sp LEB-18. Later, S. obliquus and Spirulina sp. LEB-18 were studied in a serial FBRs system, at different concentration of CO2, SO2, NO and different temperatures. The average maximum cell concentration obtained was 3,29g.L- 1 and maximum CO2 fixation 35,87%, both results for Spirulina sp. Following the CO2 biofixation study by microalgae, the strains Synechococcus nidulans and Chlorella vulgaris were isolated from Presidente Médici’s wastewater treatment station, south of Brazil. The isolated strains were cultivated and their CO2 biofixation was compared with Spirulina sp and S. obliquus. The microalgae were exposed to 12% CO2, 60ppm SO2 and 100ppm NO, simulating the flue gas. C. vulgaris showed similar behavior to that of Spirulina sp LEB-18, reaching 13,43% of maximum daily fixation. The lipid content and the fatty acids composition were determined for Spirulina sp. LEB-18, S. obliquus, S. nidulans e C. vulgaris, cultivated in a medium with 12% CO2, 60ppm SO2 and 100ppm NO at 30°C. S. obliquus showed the major lipid content (6,18%). For the other microalgae the lipid content ranged from 4,56 to 5,97%. The major AGMI content was 66,01% for S. obliquus. The PUFA were obtained in major amount by Spirulina sp. LEB-18 (29,37%) and S. nidulans (29,54%). The results showed that microalgae cultures enriched with CO2, SO2 and NO, presented a fatty acids rich biomass, being able to be used as like a nutrient source (unsaturated fatty acids), as for biofuels production (saturated fatty acids). Besides, the studied microalgae can contribute for the global warm reduction.

Picoplankton, cells between 0.2 - 2 μm, play a vital role in the carbon flow and nutrient cycling in marine food webs. Auto- and heterotrophic picoplankton dominate the biomass of oligotrophic tropical and subtropical oceans. However, little is known about their vertical distribution, changes in space and time and their relationships with environmental variables in the central Red Sea. The goal of this Ph.D. dissertation is to obtain baseline knowledge about their abundance, cellular characteristics (cell size, relative pigment and nucleic acid content) and biomass at seasonal and high-frequency temporal resolution (every 2 hours). This dissertation also aims at assessing picoplankton responses to separate and joint effects of nutrients additions (inorganic, organic and mixed) and temperature in order to be able to predict the relative contribution of eutrophication and warming in the future standing stocks of picoplankton in the Red Sea. I conducted a total of 63 vertical profiles (15 at around noon plus 48 more from the high-frequency diel samplings) from the surface down to the bottom (ca. 700 m) at a station situated 6 km off the coast of King Abdullah Economic City (KAEC) in the central Red Sea and performed 4 nutrient and temperature experiments lasting each 6 days with surface waters from the harbor of King Abdullah University of Science and Technology (KAUST). Flow cytometry allowed me to consistently identify five groups of autotrophs (Prochlorococcus, two populations of Synechococcus separated by their relative phycoerythrin fluorescence, and two differently-sized groups of picoeukaryotes) and two groups of heterotrophic prokaryotes characterized by their different relative nucleic acid content. One of the most surprising findings is the relatively lower abundances and to a lesser extent also growth rates of picoplankton compared with other tropical and subtropical oceans. Seasonality in environmental conditions emerged as an important factor in the response of picoplankton to nutrient additions and temperature. Picoplankton mostly responded to inorganic and mixed nutrient additions rather than warming. Overall, the information provided in this dissertation fills the gap of a critical component of Red Sea pelagic ecosystems and expands the information available on picoplankton communities in tropical waters.

<p>  </p> <p>Cyanobacteria are among the simplest organisms to display circadian rhythms that synchronize endogenous physiological activities with a ~12-hour-light:12-hour-dark (12L:12D) cycle of the external environment. Detected by the input pathway composed of CikA and LdpA proteins, light is transduced to the central circadian oscillator encoded by the gene cluster <em>kaiABC. </em>While KaiC phosphorylation is primarily regulated by KaiA and KaiB proteins, two key components of the output pathway, RpaA and SasA proteins, mediate between KaiC phosphorylation, genome-wide expression, and control of cell division. In this study, <em>Synechococcus elongatus </em>UTEX 2973 showed similar growth patterns when subjected to white light only and white light supplemented with ultraviolet A (UV-A) radiation under 12L:12D intervals, although UV-A radiation hindered growth during light periods. Under continuous illumination, growth rates of <em>S. elongatus </em>UTEX 2973 were reduced by UV-A radiation but reflected intrinsic circadian rhythmicity. To elucidate the critical role of the circadian clock, a mutant void of <em>kaiABC</em> was generated via the CRISPR/Cpf1 system. A dysfunctional clock severely disrupted inherent growth rhythmicity, which was exacerbated by UV-A radiation. To investigate the effects of UV-A radiation on transcription patterns in <em>S. elongatus </em>UTEX 2973, expression levels of circadian genes, specifically <em>kaiABC</em>, <em>cikA</em>, <em>lpdA</em>, <em>rpaA</em>, and <em>sasA</em>, were assessed by qPCR analysis. For the UV-A-treated wild-type strain, <em>kaiA</em> and <em>kaiB</em> expression was generally downregulated, <em>kaiC</em> expression was upregulated during the second dark period, and <em>rpaA</em> expression was either upregulated or downregulated depending on the period. For the UV-A-treated Δ<em>kaiABC </em>strain, <em>lpaA</em> expression was upregulated in darkness, whereas <em>rpaA</em> and <em>sasA</em> expression was downregulated during light periods. When Δ<em>kaiABC </em>and wild-type strains were examined in the presence and absence of UV-A radiation, expression of <em>lpaA</em>, <em>rpaA</em>, and <em>sasA</em> was universally downregulated, yet <em>cikA</em> expression was upregulated in the dark. This study was the first to evaluate the impact of UV-A radiation on cyanobacterial circadian rhythms, in which UV-A radiation negatively affected cyanobacterial growth and strongly altered gene expression patterns over time. Without the circadian clock, rhythmicity of growth and transcription was demolished, such that the consequences were aggravated for the output pathway that relayed signals downstream from the central oscillator. </p>

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Las cianobacterias, microorganismos que realizan la fotosíntesis oxigénica, tienen que adaptar su metabolismo a los distintos retos ambientales a los que se enfrentan, como la limitación de nutrientes o los ciclos de luz oscuridad. Para ello, han desarrolado una serie de mecanismos de gran complejidad y alta regulación que les permiten adaptarse y sobrevivir. En este contexto, PipX, una pequeña proteína exclusiva de cianobacterias, descubierta por el grupo de investigación de genética cianobacteriana de la Universidad de Alicante, actúa como conexión, dependiente del estatus carbono/nitrógeno, entre la proteína de transducción de señales, PII, y el regulador transcripcional, NtcA. Recientemente se han descubierto otras parejas de interacción de PipX, entre las que destaca PipY, miembro de la familia de proteínas de unión a piridoxal fosfato (PLPBP) que forma un operón con PipX en la mayoría de cianobacterias, y la GTPasa de ensamblaje de ribosomas, EngA. La mayoría de estos descubrimientos se han realizado en el organismo modelo, Synechococcus elongatus PCC7942, el cual ha sido el principal objeto de estudio en esta Tesis, centrada fundamentalmente en PipX y sus parejas de interacción. Entre las aportaciones novedosas de estas Tesis se encuentran: 1. La propuesta del empleo de PipY como modelo para el estudio de miembros de la familia PLPBP/COG325; 2. La caracterización de los fenotipos de sobreexpresión de PipX y PipY, dando lugar al descubrimiento de nuevas funciones (formación de polifosfatos) y conexiones entre ellas; 3. La demostración de la existencia de interacción funcional entre EngA y PipX, y la descripción de la función de EngA en el estrés redox en cianobacterias; 4. El avance en el estudio de los terminantes moleculares de la toxicidad de PipX en ausencia de PII y el papel de esta última en el mantenimiento de los niveles intracelulares de PipX. En conclusión, esta Tesis amplía el conocimiento sobre la compleja regulación de los sistemas cianobacterianos en respuesta a distintos estímulos ambientales y, en concreto, las conexiones y el papel de PipX junto a sus antiguas y nuevas parejas de interacción.