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Diatoms are crucially important to the global ecosystem due to their role in regulating the world’s carbon and silicon cycles, and their production of large amounts of organic material in aquatic environments. They are thought to comprise ca. 100,000 species, although some estimates suggest that there could be over a million. Despite their importance and high species diversity, little is known about their phylogeny due to technical issues that hinder the reconstruction of their relationships. However, owing to a new technique that allows for DNA to be amplified from a single isolated cell, it is possible to explore diatom relationships with extensive taxonomic sampling. This thesis aims to demonstrate that the integration of molecular data and morphological characters can provide a new paradigm for future phylogenetic and taxonomic studies of diatoms, especially among closely related and taxonomically complex groups. To achieve this, I examined common species from two naviculoid diatom genera, Frustulia and Navicula using sequence data from three molecular markers (rbcL, atpB, 18S), traditional and fine-scale morphological characters, and frustule shape. The molecular markers rbcL and atpB evolved at a similar rate and performed well at reconstructing species-level phylogenies, whereas 18S was more conserved and best used for resolving relationships at higher taxonomic levels. Hidden diversity was uncovered in what have traditionally been thought as well-circumscribed taxa, and three new species were described. The methods used here show promise for the future of diatom systematics.

El biodiesel se obtiene a nivel industrial, casi en su totalidad, a partir de aceites de semillas. Sin embargo, el incremento de la demanda energética, el uso del agua dulce y la utilización de semillas con fines alimenticios plantean la necesidad y búsqueda de nuevas fuentes de materias primas sustentables que, además, posean impactos mínimos sobre el medio ambiente. Muchas especies de microalgas, denominadas oleaginosas, han despertado gran interés como fuente potencial de lípidos neutros (LN), específicamente triglicéridos (TAG), considerados la materia prima ideal para la producción de biodiesel. Una posible estrategia para mejorar los costos de producción del biodiesel de tercera generación es la búsqueda y selección de especies nativas de microalgas que, además de producir TAG, sean capaces de sintetizar productos de alto valor agregado. Sobre la base de estos antecedentes, el objetivo general de esta tesis fue evaluar el potencial de una especie de diatomea nativa del Estuario de Bahía Blanca como materia prima para la producción de biodiesel y sustancias poliméricas extracelulares como co-productos de valor agregado, bajo un concepto de biorrefinería. Los objetivos específicos planteados fueron: 1) la identificación de la especie; 2) la optimización de técnicas para la determinación de la densidad celular, la concentración de pigmentos fotosintéticos y el contenido de lípidos neutros en cultivos a escala de laboratorio; 3) analizar el efecto de distintas condiciones ambientales (temperatura [15ºC y 20ºC], medios de cultivo [f/2, SWES, y SWES-N] y la fase estacionaria sobre parámetros de crecimiento, producción de biomasa, cantidad y calidad de lípidos y productividad lipídica con especial énfasis en la fracción de lípidos neutros; 4) evaluar el potencial de las sustancias poliméricas extracelulares liberadas por N. cincta como co-producto de valor; y 5) analizar el crecimiento de la especie en un fotobiorreactor columnar de 25L. Los estudios morfológicos con microscopios óptico y electrónico de barrido permitieron identificar a la especie en estudio como Navicula cincta, una diatomea pennada. La cepa fue depositada en el Culture Collection of Autotrophic Organism (CCALA), República Checa, bajo la denominación Navicula cincta CCALA 984. La velocidad de crecimiento de N. cincta varió entre 0,29 div. día-1 y 1,42 div. día-1, mientras que el tiempo de generación varió entre 16,8 horas y 82,5 horas. Las máximas densidades celulares (DC) fueron obtenidas con el medio SWESN. Se estableció un modelo entre la DC y la absorbancia, el cual permitió monitorear el crecimiento de N. cincta en f/2. La espectrofluorometría de la clorofila a se presentó como un método adecuado para evaluar la cinética de crecimiento y para obtener información sobre el estado fisiológico de los cultivos. La cinética de clorofila a in vivo presentó la misma dinámica que la de la DC en los tres medios de cultivo testeados. El medio f/2 resultó adecuado para producir un rápido crecimiento seguido de una fase estacionaria temprana, mientras que los medios SWES y SWES-N permitieron un crecimiento sostenido a lo largo de todo el período considerado. La relación IFR-Cel establecida entre la intensidad de fluorescencia relativa del cultivo teñido con Rojo Nilo (IFR-RN) y la DC se propone como un índice para estimar el tiempo de acumulación máxima de LN y, por lo tanto, determinar el tiempo de cosecha de la biomasa. Esta información resultó útil para cultivos llevados a cabo en medio f/2, pero no así en los desarrollados en los medios SWES y SWES-N, debido a que en éstos últimos no se detectó acumulación lipídica. El contenido de lípidos totales (LT) de N. cincta varió entre 6-40 % del peso seco libre de cenizas (% PSLC). En las condiciones de crecimiento evaluadas, los LN o TAG representaron entre un 50 % y 90% de los LT. La mayor productividad de LN fue de 10 mg. L-1. d-1 en los cultivos creciendo a 20°C en f/2 durante 10 días. Los valores más bajos de LT y LN se obtuvieron con los medios SWES y SWES-N, sugiriendo que en estos medios no hubo acumulación lipídica. Con respecto a la composición de los LN, los ácidos grasos monoinsaturados (AGMI) fueron la clase dominante representando el 53% a 60%, seguida por los ácidos grasos saturados (AGS) que constituyeron entre el 22% a 28%. Una excepción a este patrón ocurrió en los cultivos en SWES-N, donde los contenidos de AGMI y AGS de la fracción neutra fueron similares (39% y 38%, respectivamente). Los ácidos grasos poliinsaturados (AGPI) fueron la fracción minoritaria en todos los casos testeados. El perfil de ácidos grasos de la fracción neutra fue similar en todas las condiciones de cultivo testeadas. Los principales ácidos grasos fueron los ácidos palmítico (16:0), palmitoleico (16:1 (n-7)) y eicosapentaenoico (20:5 (n-3)). En relación a los parámetros de calidad del aceite, el índice de Yodo y el contenido de ácido linolénico estuvieron dentro de los límites establecidos por el estándar de calidad europeo EN14214. Estas características permiten inferir que los aceites de N. cincta se presentan como una materia prima adecuada para la producción de biodiesel. En las cinéticas de nutrientes se pudo observar una limitación de fósforo y sílice en el medio f/2, durante la fase estacionaria. En los medios SWES y SWES-N, los nutrientes no alcanzaron valores limitantes para el crecimiento, lo cual se visualizó en un incremento sostenido en la DC y en la baja acumulación de LN. Las sustancias poliméricas extracelulares (EPS) de N. cincta fueron clasificadas en EPS solubles (EPS-S), EPS levemente unidos a la célula (EPSLU) y EPS fuertemente unidos (EPS-FU) a la célula. Las cinéticas de producción de las mismas fueron analizadas en los tres medios de cultivo. Los EPS-S fueron el principal constituyente en los medios f/2 y SWES (77% y 86% de los EPS totales, respectivamente), mientras que en SWES-N representaron la tercera parte del total de EPS. La producción media de los EPS-S estaría relacionada con las relaciones estequiométricas N/P. En general, los EPS-FU fueron más abundantes que los EPS-LU y esto podría estar asociado al hábito bentónico de la especie. La composición de monosacáridos neutros de los EPS-S fue similar a la de los EPS-FU. Sus principales componentes fueron xilosa, manosa y N-acetilglucosamina. Los EPS-LU estuvieron mayormente constituidos por galactosa y glucosa. La relación carbohidrato:proteína y la identificación de la N-acetilglucosamina y la N-acetilgalactosamina sugieren la presencia de proteoglucanos y/o glicoproteínas en los EPS. Navicula cincta fue cultivada en un fotobiorreactor columnar con medio SWESN y con CO2. Los cultivos presentaron una fase exponencial, estacionaria y una de disminución celular, no detectada a escala de laboratorio. La producción de biomasa fue de 319 mg. L-1. La cinética espectrofluorométrica del Rojo Nilo evidenció acumulación de LN durante la fase estacionaria. El contenido de LT fue de 32 % PSLC, representando los LN un 81 % de los LT. La productividad de los LN fue de 51 mg. L-1.d-1. Con respecto a la composición de ácidos grasos de los LN, el porcentaje de AGMI fue de 51%, mientras que los de AGS y AGPI fueron de 32% y 16%, respectivamente. Los principales ácidos grasos fueron los ácidos palmítico, palmitoleico y eicosapentaenoico. De acuerdo con los resultados obtenidos, se puede concluir que la microalga oleaginosa Navicula cincta puede ser considerada como una especie potencialmente útil para la producción de biodiesel. Además, la producción simultánea de TAG para biodiesel y de EPS como co-producto, bajo un contexto de biorrefinería, se presenta como una propuesta novedosa para ser aplicada en cultivos energéticos sustentables. / Biodiesel is obtained at industrial level almost entirely from seed oils. However, factors like increasing of the demand of energy, freshwater use and the utilization of seeds for food purposes suggest the need and search for new sources of sustainable and alternative feedstocks, which have minimal impacts on the environment. Many species of microalgae, called oleaginous, have raised great interest as a potential source of neutral lipids, specifically triglycerides (TAG), which are considered the ideal feedstock for biodiesel production. A possible strategy for improving the cost of production of thirdgeneration biodiesel is the search and selection of microalgae native species that can produce TAG and could be able to synthesize valuable co-products. On the basis of previous background, the objective of this thesis was to evaluate the potential of a native diatom from Bahía Blanca Estuary as raw material for both biodiesel production and extracellular polymeric substances as valuable co-products, under a concept of biorefinery. The specific objectives were: 1) to identify the species; 2) to optimize different techniques for the determination of cell density, the photosynthetic pigment concentration and the content of neutral lipids in cultures at laboratory scale; 3) to analyse the effect of different environmental conditions (temperature [15°C and 20°C], culture media [f/2, SWES, SWES-N] and stationary phase) on growth parameters, biomass production, lipid quantity and quality and lipid productivity, with particular emphasis on neutral lipid fraction; 4) to evaluate of the potential of the extracellular polymeric substances released by N. cincta as an added value coproduct; 5) to analyse the growth of the species in a 25L columnar photobioreactor. Both morphological studies with optical and scanning electronic microscopes allowed identifying the studied strain as Navicula cincta, a pennate diatom. The strain was deposited in the Culture Collection of Autotrophic Organism (CCALA), Czech Republic, under the name Navicula cincta CCALA 984. The growth rate of N. cincta ranged between 0,29 div. día-1 and 1,42 div. día-1, while the time of generation ranged between 16,8 hours y 82,5 hours. The maximum cell densities (CD) were obtained with the SWES-N medium. A model between the CD and the absorbance was established. It allowed monitoring the growth of N. cincta in f/2. The spectrofluorometry of chlorophyll a was presented as an adequate method to evaluate the kinetic of growth and to give information about the physiological state of the cultures. The kinetic of the chlorophyll a in vivo presented the same dynamic as the CD in the three culture media tested.The f/2 medium was suitable for producing a rapid growth followed by an early stationary phase. Moreover SWES and SWES-N media allowed a sustained growth along the all considered period. The relationship IFR-Cel established between the relative fluorescence intensity of Nile Red dyed culture (NR-RFI) and the DC was proposed as an index to estimate the maximum neutral lipids accumulation time and, consequently, to determine the biomass harvesting time. This information was useful for cultures performed in f/2 medium, but this index was not useful for the culture developed in SWES or SWES-N media, because in these media lipid accumulation was not detected. The total lipid content (TL) of N. cincta varied between 6- 40% of ash-free dry weight (% of AFDW). Under growing conditions evaluated, the NL or TAG represented between 50% and 90% of TL. The highest NL productivity was 10 mg. L-1.d-1 in culture growing at 20ºC in f/2 medium for 10 days. The lowest values of TL and NL were obtained with SWES-N and SWES, suggesting that, in these culture media, there was not lipid accumulation. Respect of the composition of LN, monounsaturated fatty acids (MUFA) were the dominant class representing from 53% to 60%, followed by saturated fatty acids (SFA) that they constituted from 21% to 28%. An exception of this pattern occurred in SWES-N culture, where the contents of MUFA and SFA of NL were similar (39% and 38%, respectively). Polyunsaturated fatty acids (PUFA) were the minority fraction in all tested cases. The fatty acid profile of the neutral fraction was similar under all evaluated culture conditions. The main fatty acids were the acids palmitic (16:0), palmitoleic (16:1 (n-7)) and eicosapentaenoic (20:5 (n-3)). Regarding to oil quality parameters, iodine index and content of linolenic acid were within the limits established by European quality standard EN14214. These features allowed inferring that the oils of N. cincta constitute a suitable feedstock for biodiesel production. In the kinetics of nutrients could be observed a limitation of phosphate and silicate in f/2 medium, during early stationary phase. In the SWES and SWES-N media, nutrients did not reach limiting values for growth, which was visualized in a sustained increase of DC and lower accumulation of LN. Extracellular polymeric substances (EPS) of N. cincta were classified into soluble EPS (S-EPS), cell loosely bound EPS (LB-EPS) and cell tightly bound EPS (TB-EPS). The kinetic of production of these was analyzed in the three culture media. The EPS-S was the major component in both f/2 and SWES media (77% and 86% of the total EPS, respectively), while in SWES-N it was a third of the total EPS. The average production of EPS-S would be related to stoichiometric ratios N / P. In general, the TB-EPS were more abundant than the LB-EPS and this could be associated with the benthic habit of the species. The S-EPS and TB-EPS neutral monosaccharide composition was similar. Their main components were xylose, mannose and N-acetylglucosamine. The LB-EPS was largely constituted by galactose and glucose. The carbohydrate:protein ratio as well as the identification of N-acetylgalactosamine and N-acetylglucosamine suggest the presence of proteoglycans and/or glycoproteins in the EPS. Navicula cincta was grown in a columnar photobioreactor with SWES-N and CO2. The culture exhibited exponential phase, stationary phase and cell decreased phase that was not detected at laboratory scale. Biomass production was 319 mg. L-1. The kinetic of the spectrofluorometry of Nile Red showed accumulation of LN during stationary phase. The content of TL was 32 (% AFDW), being the NL 81% of TL. The NL productivity was 51 mg. L-1.d-1. In regard to fatty acid composition of NL, MUFA percentage was 51%, while the SFA and PUFA were 32% and 16%, respectively. The main fatty acids were the acids palmitic, palmitoleic and eicosapentaenoic. According to the obtained results, it is concluded that the oleaginous microalgae Navicula cincta can be considered as a potentially useful species for production of biodiesel. In addition, the simultaneous production of TAG for biodiesel and EPS as co-product, under a biorefinery context, is presented as a novel proposal to be applied in sustainable energetic cultures.

Submitted by Jennifer Paola Moyón Dávila (jennifermoyon05@gmail.com) on 2016-09-23T00:30:13Z No. of bitstreams: 1 Dissertação Jennifer Moyon Davila.pdf: 5166920 bytes, checksum: cea59c17ed8b8c782f55743ec0a9b278 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-09-26T18:49:25Z (GMT) No. of bitstreams: 1 davila_jpm_me_rcla.pdf: 5166920 bytes, checksum: cea59c17ed8b8c782f55743ec0a9b278 (MD5) / Made available in DSpace on 2016-09-26T18:49:25Z (GMT). No. of bitstreams: 1 davila_jpm_me_rcla.pdf: 5166920 bytes, checksum: cea59c17ed8b8c782f55743ec0a9b278 (MD5) Previous issue date: 2016-08-04 / O gênero Navicula está classificado na Família Naviculaceae e inclui, em seu ‘sensu stricto’, entre 250 e 300 espécies habitantes das águas doces e marinhas, que se caracterizam, principalmente, pela presença de estrias radiadas na região mediana da valva e pelas terminações proximais da rafe desviadas para o lado primário ou secundário da valva, onde se localiza a falha de Voigt. Em alguns casos, as terminações proximais da rafe são retas. Não existe publicação de cunho taxonômico exclusivo sobre representantes de Navicula realizada para o Estado de São Paulo. Por esta razão, o presente estudo visou a aprofundar o conhecimento taxonômico das espécies de Navicula ‘sensu stricto’ com base no estudo de populações amostradas de 32 reservatórios distribuídos em cinco bacias hidrográficas do Estado de São Paulo. O presente estudo foi realizado a partir da análise de 317 lâminas permanentes que abrangeram hábitats como fitoplâncton, perifíton e sedimentos superficiais. O material planctônico foi coletado com amostrador de van Dorn e rede de plâncton confeccionada com tecido de náilon de 20 μm de abertura de malha. O material perifítico foi recolhido a partir de substratos naturais (rochas ou macrófitas aquáticas) e o dos sedimentos superficiais foi coletado com testemunhador de gravidade, aproveitando os dois primeiros centímetros superficiais. Todas as amostras foram fixadas com solução aquosa de formalina a 3-5% e, em seguida, oxidadas e analisadas ao microscópio óptico. Foram identificadas 38 espécies e dois materiais em nível gênero; e todos foram descritos, ilustrados e comentados. Características morfológicas e métricas foram incluídas para todos os 40 táxons. Nenhum táxon teve ocorrência igual ou superior a 50% nas amostras analisadas, sendo Navicula notha (23% das amostras analisadas) a espécie que apresentou a maior frequência de ocorrência, seguida de N. cryptocephala (14,2%), N. rostellata (9,8%), N. neomundana (7,6%), N. breintenbuchii (6,6%), N. cryptotenella (5,4%), N. herbstiae (4,1%), N. symmetrica (3,8%) e N. radiosa (2,5%). Vinte e uma espécies foram identificadas pioneiramente para o Estado de São Paulo. Por fim, o presente estudo contribuiu para o conhecimento da biodiversidade de diatomáceas do Estado de São Paulo e o fornecimento de informação fundamental para outros estudos taxonômicos, ecológicos e demais que demandem a identificação adequada da alga estudada. / Genus Navicula is classified in the Family Naviculaceae and includes in its ‘sensu stricto’ between 250 and 300 species living in the fresh and marine waters, that are characteristic, mainly by the presence of radiating striae at the median portion of valves and the raphe proximal endings deviating to the valve primary or secondary side, where is located the Voigt discontinuity. In some instances, however, raphe proximal endings are straight. There is no publication dealing exclusively with the taxonomy of Navicula representatives for São Paulo State. For this reason, present study aimed at deepening the taxonomical knowledge of Navicula ‘sensu stricto’ species based on the study of populations sampled from 32 reservoirs distributed among five water basins of São Paulo State. Present study was based on the analyses of 317 permanent mountings that involved diferente habitats (phytoplankton, periphyton and surface sediments). Plankton material was gathered with a van Dorn sampler and a net built of a 20 μm mesh nylon fabric. Periphytic material was collected from natural substrates (rocks or aquatic macrophytes), and that of surface sediments by using a gravity corer using just the first two superficial centimeters. All samples were fixed and preserved with a formalin 3-5% water solution, and immediately after oxidated and observed under a light microscope. Thirty eight species were identified and two populations just to the genus level; all 40 materials were described, illustrated and commented. Morphological and metric characteristics were made available for all 40 taxa. No taxon had its occurrence ≥ 50% of total samples examined, Navicula notha (23% of samples analysed) being the species that showed the highest occurrence frequence, followed by N. cryptocephala (14.2%), N. rostellata (9.8%), N. neomundana (7.6%), N. breintenbuchii (6.6%), N. cryptotenella (5.4%), N. herbstiae (4.1%), N. symmetrica (3.8%) and N. radiosa (2.5%). Twenty one species were presently identified for the first time for São Paulo State. Finally, present study contributes to the knowledge of the diatoms biodiversity of São Paulo State, and provides fundamental information to other taxonomic, ecological, and other studies that will need the proper taxonomic identification of the alga studied.

<p>Benthic diatoms are valuable tools for biological monitoring and paleo-ecological reconstruction of past environmental conditions. This thesis aims at describing size-related properties of benthic diatoms and suggests that data assessment for community analysis can be improved by considering the importance of scale. It investigates which environmental factors structure epilithic diatom communities on the coast of the Gulf of Bothnia and identifies environmental factors correlated with phytobenthic biomass. It also contributes to the floristic knowledge of the Baltic Sea. </p><p>Diatom species show large variation in size. The responses of large species (≥1000 μm3) in diatom communities to environmental factors are underestimated if solely measured as relative abundance, as is the common practice. However, relative abundance gives the best gradient resolution, as compared to surface area and biovolume, if species are counted separately in two biovolume classes. Small and large species in the same community may respond differently to the same environmental factors. </p><p>To assess the principal environmental factors structuring diatom communities in the Gulf of Bothnia, 270 quantitative samples were collected from submerged stones. Sampling was carried out in spring in four areas of the Bothnian Bay, characterized by a stable north-south salinity gradient (0.4-3.3 psu), and in three areas of the Bothnian Sea which has a rather uniform salinity of ca. 5 psu. Canonical correspondence analysis (CCA) identified salinity and exposure to wave action as the principal factors structuring the diatom communities of the Bothnian Bay, whereas exposure to wave action was the principal factor in the Bothnian Sea. Measurements of relative ignition loss suggested that the cover of macroalgae, and thereby the higher abundance of epiphytic diatoms in the epilithic samples, was positively correlated with salinity in the Bothnian Bay and with water movement in the Bothnian Sea. </p><p>Two new brackish water species are described, <i>Navicula sjoersii</i> S. Busse & Snoeijs and <i>N. bossvikensis</i> S. Busse & Snoeijs. The new species are compared with <i>N. perminuta</i> Grunow, a common brackish-water species.</p>

Benthic diatoms are valuable tools for biological monitoring and paleo-ecological reconstruction of past environmental conditions. This thesis aims at describing size-related properties of benthic diatoms and suggests that data assessment for community analysis can be improved by considering the importance of scale. It investigates which environmental factors structure epilithic diatom communities on the coast of the Gulf of Bothnia and identifies environmental factors correlated with phytobenthic biomass. It also contributes to the floristic knowledge of the Baltic Sea. Diatom species show large variation in size. The responses of large species (≥1000 μm3) in diatom communities to environmental factors are underestimated if solely measured as relative abundance, as is the common practice. However, relative abundance gives the best gradient resolution, as compared to surface area and biovolume, if species are counted separately in two biovolume classes. Small and large species in the same community may respond differently to the same environmental factors. To assess the principal environmental factors structuring diatom communities in the Gulf of Bothnia, 270 quantitative samples were collected from submerged stones. Sampling was carried out in spring in four areas of the Bothnian Bay, characterized by a stable north-south salinity gradient (0.4-3.3 psu), and in three areas of the Bothnian Sea which has a rather uniform salinity of ca. 5 psu. Canonical correspondence analysis (CCA) identified salinity and exposure to wave action as the principal factors structuring the diatom communities of the Bothnian Bay, whereas exposure to wave action was the principal factor in the Bothnian Sea. Measurements of relative ignition loss suggested that the cover of macroalgae, and thereby the higher abundance of epiphytic diatoms in the epilithic samples, was positively correlated with salinity in the Bothnian Bay and with water movement in the Bothnian Sea. Two new brackish water species are described, Navicula sjoersii S. Busse &amp; Snoeijs and N. bossvikensis S. Busse &amp; Snoeijs. The new species are compared with N. perminuta Grunow, a common brackish-water species.