Dissertationen zum Thema „Culture heterotrophe“

Les microalgues sont considérées comme une solution biotechnologique aux problèmesmajeurs de ce siècle, offrant des réponses et des alternatives dans des domainestels que la malnutrition, les pénuries alimentaires, le changement climatique etla pollution. En tant qu'organismes photosynthétiques, les microalgues synthétisent denombreux composés afin d'exploiter la lumière du soleil pour leurs fonctions métaboliques, notamment la lutéine, un caroténoïde jaune essentiel pour l'alimentation humaine.Bien que la lutéine soit un composé associé à l'activité photosynthétique des microalgues,certaines espèces conservent la capacité de synthétiser cet important pigmentmême dans des conditions de culture hétérotrophe, ce qui permet d'augmenter la productivité de la biomasse et de la lutéine. Scenedesmus almeriensis a été rapportée comme une bonne productrice de lutéine dans des conditions phototrophiques, mais son faible taux de croissance par rapport à d'autres espèces de microalgues a entravé son potentiel.Cette thèse présente les résultats de la productivité en lutéine obtenue à partir d'uneculture hétérotrophe de Scenedesmus almeriensis, à la fois à l'échelle du laboratoire et àl'échelle pré-pilote. En plus d'atteindre des valeurs élevées de productivité en lutéine, sataille cellulaire plus grande et sa fragilité simplifient la récolte de la biomasse et l'extraction des pigments. Une méthode simplifiée d'extraction de la lutéine adaptée à cette espèce est également présentée. Ce procédé utilise l'éthanol comme solvant d'extraction et nécessite moins de temps et d'énergie, ce qui représente des avantages pour l'environnement et l'acceptation dans l'industrie
Microalgae are positioned as a biotechnological solution to major problems ofthis century, offering answers and alternatives in areas like malnutrition, foodshortages, climate change, and pollution. As photosynthetic organisms, microalgaesynthesize numerous compounds to harness sunlight for their metabolic functions,including lutein, a yellow carotenoid crucial for the human diet.Although lutein is a compound associated with the photosynthetic activity of microalgae,some species maintain the capacity to synthesize this important pigment even underheterotrophic conditions, allowing increased productivity of both biomass and lutein incultures supplemented with an organic carbon source. Scenedesmus almeriensis has been reported as a good lutein producer under phototrophic conditions, but its low growth rate compared to other microalgae species has hindered its potential.This thesis presents results of the lutein productivity obteained from a heterotrophicculture of Scenedesmus almeriensis, both at laboratory and pre-pilot scale. In addition toachieving high lutein productivity values, its larger cell size and fragility simplify biomassharvesting and pigment extraction. A simplified method for lutein extraction adaptedto this species is also presented. This process uses ethanol as an extraction solvent andrequires less time and energy, which represents environmental and product acceptanceadvantages

Els oceans conten aproximadament un total de 10^29 cèl·lules microbianes. Els bacteris marins són responsables de la major part de la respiració que es produeix en l’oceà i són essencials en els cicles biogeoquímics de la Terra. Estudiar la diversitat bacteriana dels ecosistemes marins i tenir accés als genomes mitjançant estudis dependents o independents de cultiu és important per desxifrar el potencial metabòlic dels bacteris marins. El cultius ens aporten informació sobre la fisiologia bacteriana, ecologia i contingut genòmic, però la majoria dels esforços en aïllar bacteris marins provenen de la zona fòtica de l’oceà, deixant les profunditats marines menys explorades. En aquesta tesi, tècniques estàndard de cultiu han permès crear una col·lecció marina de cultius de bacteris heterotròfics (MARINHET), composada per més de 2000 aïllats, recuperats de diverses regions oceanogràfiques, de diverses profunditats (superfície, mesopelàgic i batipelàgic) i cobrint diverses estacions i anys. El Capítol 1 descriu la seva taxonomia, diversitat filogenètica i biogeografia i revela que un 37% de les soques aïllades són 100% idèntiques en el gen ribosomal 16S (16S rRNA) entre la zona fòtica (superfície) i afòtica (mesopelàgic i batipelàgic). A més, hem identificat Alteromonas i Erythrobacter entre els gèneres marins heterotròfics més comuns que recuperem en cultiu usant un medi marí estàndard. Les tècniques tradicionals de cultiu generalment només recuperen una fracció petita de les comunitats bacterianes naturals, fenomen conegut com ‘la gran anomalia de recompte en placa’ i moltes de les soques que s’aïllen pertanyen a la biosfera rara. Tanmateix, no coneixem si aquests patrons, normalment descrits per als bacteris de la zona fòtica, també s’apliquen als bacteris de les profunditats. En el Capítol 2 he combinat els resultats obtinguts mitjançant tècniques dependents i independents de cultiu comparant les seqüències del 16S rRNA de la col·lecció MARINHET contra fragments de seqüenciació massiva del 16S rRNA, tant d’amplicons com de metagenomes, obtinguts de mostres globalment distribuïdes i de diferents profunditats. Trobem que una major proporció dels bacteris de l’oceà profund són cultivables i una fracció important dels aïllats té preferència a un estil de vida adherit a partícules. A més, confirmem que el dogma ‘menys de l’1% dels bacteris són cultivables’ ha de ser revisat ja que trobem variabilitat en les mostres de l’oceà profund, on fins a un 3% de les cèl·lules han sigut cultivades. Els aïllats bacterians són un excel·lent material per a aplicacions biotecnològiques com la bioremediació de zones marines contaminades. El mercuri és un metall pesat tòxic i la seva forma més perillosa, el metilmercuri (MeHg), es bioacumula a la cadena tròfica marina. No obstant això, es coneix molt poc la tolerància de bacteris marins enfront del mercuri o la fisiologia d’aquelles soques que codifiquen l’operó dels gens de resistència (operó mer). El Capítol 3 descriu els resultats del mapeig funcional dels gens merA i merB, clau en la detoxificació, en una fracció de la col·lecció MARINHET. Ens centrem en dos generes marins, amb un potencial genètic per a la degradació del mercuri conegut, com són Alteromonas i Marinobacter. Revelem que els gens merAB estan àmpliament distribuïts en diferents regions oceanogràfiques i en diverses profunditats. Addicionalment, hem seleccionat una soca d’ Alteromonas mediterranea per a futurs estudis de bioremediació degut a la seva alta tolerància i capacitat de degradació de diferents formes de mercuri.
Los océanos contienen aproximadamente un total de 10^29 células microbianas. Las bacterias marinas son responsables de la mayor parte de la respiración que se produce en el océano y son esenciales en los ciclos biogeoquímicos de la Tierra. Estudiar la diversidad bacteriana de los ecosistemas marinos y tener acceso a los genomas mediante estudios dependientes e independientes de cultivo es importante para descifrar el potencial metabólico de las bacterias marinas. Los cultivos nos aportan información sobre la fisiología bacteriana, ecología y contenido genómico, pero la mayoría de los esfuerzos en aislar bacteria marinas provienen de la zona fótica del océano, dejando las profundidades marinas menos exploradas. En esta tesis, técnicas estándar de cultivo han permitido crear una colección marina de bacterias heterótrofas (MARINHET), compuesta por más de 2000 aislados, recuperados de varias regiones oceanográficas, de varias profundidades (superficie, mesopelágico y batipelágico), y cubriendo varias estaciones y años. El Capítulo 1 describe su taxonomía, diversidad filogenética y biogeografía y revela que un 37% de las cepas son 100% idénticas en la secuencia parcial del gen ribosomal 16S (16S rRNA) entre la zona fótica (superficie) y afótica (mesopelágico y batipelágico). Además, hemos identificado Alteromonas y Erythrobacter entre los géneros marinos heterótrofos más comunes que recuperamos en cultivo usando un medio marino estándar. Las técnicas tradicionales de cultivo generalmente solo recuperan una fracción pequeña de las comunidades bacterianas naturales, fenómeno conocido como ‘la gran anomalía de recuento en placa’ y muchas de las cepas que se aíslan pertenecen a la biosfera rara. Sin embargo, no conocemos si estos patrones, normalmente descritos para las bacterias de superficie, también se aplican en las profundidades. En el Capítulo 2 he combinado resultados obtenidos mediante técnicas dependientes e independientes de cultivo comparando las secuencias del 16S rRNA de la colección MARINHET contra los fragmentos de secuenciación masiva del 16S rRNA (de amplicones y metagenomas), obtenidos de muestras globalmente distribuidas y de diferentes profundidades. Una mayor proporción de las bacterias del océano profundo son cultivables y una fracción importante de los aislados tiene preferencia a un estilo de vida adherido a partículas. Además, confirmamos que el dogma ‘menos del 1% de las bacterias son cultivables’ deber ser revisado ya que encontramos variabilidad en las muestras de profundidad, donde hasta un 3% de las células se han podido aislar. Los aislados bacterianos son un excelente material para aplicaciones biotecnológicas, como la biorremediación de zonas marinas contaminadas. El mercurio es un metal pesado tóxico y su forma más peligrosa, el metilmercurio (MeHg), se bioacumula en la cadena trófica marina. No obstante, se conoce muy poco la tolerancia de bacterias marinas frente al mercurio o la fisiológia de aquellas cepas que codifican los genes de resistencia (operón mer). El Capítulo 3 describe los resultados del mapeo funcional de los genes merA y merB, clave en la detoxificación, en una fracción de la colección MARINHET. Nos centramos en dos géneros marinos, con un potencial genético para la degradación del mercurio previamente descrito en la literatura, como son Alteromonas y Marinobacter. Desvelamos que los genes merAB están ampliamente distribuidos en diferentes regiones oceanográficas y en varias profundidades. Adicionalmente, hemos seleccionado una cepa de Alteromonas mediterranea para futuros estudios de biorremediación debido a su alta tolerancia y capacidad de degradación de diferentes formas de mercurio.
The world’s oceans sustain the life for an estimated total of 10^29 microbial cells. Marine bacteria are responsible for most part of the ocean respiration and are key in most biogeochemical cycles of the Earth. Accordingly, the study of the bacterial diversity present in different marine ecosystems is essential, and having access to their genomes through isolation or genomic centric studies is important to decipher their metabolic potential. Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. In this thesis, standard plating techniques allowed to create a marine culture collection of heterotrophic bacteria (MARINHET). More than 2000 isolates were retrieved from samples collected from a variety of oceanographic regions, from different depths including surface, mesopelagic and bathypelagic waters, and also covering different seasons and years. Chapter 1 describes the taxonomy, the phylogenetic diversity and the biogeography of culturable heterotrophic marine bacteria, and reveals that an important percentage of the strains (37%) are 100% identical in their partial 16S rRNA gene between photic and aphotic layers. In addition, we identified Alteromonas and Erythrobacter genera as the most frequently retrieved heterotrophic bacteria from the ocean in standard marine agar medium. It is a long-standing observation that traditional culture techniques only retrieve a small fraction of the microbial diversity found in natural environments including marine ecosystems, what is known as ‘the great plate count anomaly’. In addition, most of the retrieved isolates belong to the so-called rare biosphere. However, we do not know if these patterns, usually described for bacteria living in the photic ocean, also apply for the deep ocean bacteria. In Chapter 2 of this thesis, I combined results from culture-dependent and -independent techniques by comparing the 16S rRNA partial sequences of the MARINHET isolates with 16S rRNA amplicon Illumina TAGs (16S iTAGs) and metagenomic TAGs (miTAGs) from surface, mesopelagic and bathypelagic samples globally distributed. A high proportion of bacteria inhabiting the deep ocean could be retrieved by pure culture techniques and a significant fraction of the isolates preferred a lifestyle attached to particles. Additionally, I revised the axiom that ‘less than 1% of bacteria can be cultured’, finding variability between mesopelagic and bathypelagic samples, where up to 3% of the cells could be cultured. Bacterial isolates also represent a valuable genetic reservoir for biotechnology applications, such as bioremediation strategies of marine polluted environments. Mercury is one of the most toxic heavy metals in the planet and its most dangerous form, methylmercury (MeHg), is being bioaccumulated in the marine food web. However, little is known about the tolerance capacity and phenotypic characterization of marine bacteria codifying the mercury resistance operon (mer operon). Chapter 3 describes the functional screening of merA and merB genes, which are key in the mercury detoxification process, in well know marine genera with described genetic potential for mercury detoxification, such as Alteromonas and Marinobacter. I reported that the merAB genes from these two genera are widely distributed in different oceanographic regions and depths. In addition, I selected a promising candidate, phylogenetically affiliated to Alteromonas mediterranea, for future bioremediation studies due to its high tolerance and degradation ability of different mercury forms.
Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologia

This thesis investigates the effects of co-culturing microorganisms including 37 yeast, 38 bacteria, nine diazotrophic cyanobacteria, and three fungi on biomass and lipid production in fresh- and saltwater algae. Algal lipid content was measured using Nile Red method and gravimetric techniques. Among the algal strains tested, freshwater Coelastrum sp. 46-4, and saltwater Cricosphaera sp. 146-2-9, showed enhanced biomass yield and lipid content in response to co-culture with bacteria, cyanobacteria, and fungi. While co-culture with yeast caused inhibition of algal productivity, no difference in algal productivity was observed between nitrogen-free diazotrophic cyanobacterial co-culture and nitrogen-replete monoalgal culture. Results indicated that extracellular compounds from the freshwater bacteria Pseudomonas stutzeri and marine fungus Fusarium sp. significantly account for stimulation of lipid accumulation within algal cells, while co-cultivation with live microorganism cells stimulated biomass production in algae.

Chlorella vulgaris (C. vulgaris) est un organisme modèle qui présente un potentiel commercial élevé dans le domaine de l'alimentation et de l'énergie, avec une faisabilité prouvée de cultures sous forme de biofilms et de co-culture de levures/microalgues pour la valorisation in situ du CO2 dans les processus biotechnologiques. Ce travail de doctorat se concentre sur les colonies immobilisées dans des cultures pures ou mixtes. Il vise une meilleure compréhension des interactions au sein des colonies et entre colonies, avec pour objectif ultime de comprendre et d'optimiser les co-cultures.Pour atteindre ces objectifs, un protocole complet et des dispositifs expérimentaux innovants ont été développés, notamment des techniques d'inoculation, des dispositifs de culture immobilisée avec capteurs de gaz, l'imagerie 3-D à l'aide d'un microscope à lumière structurée, le traitement d'images, un bilan gazeux calibré et l'analyse des données. Des précautions ont également été prises concernant les conditions d'incubation, la détermination de la masse sèche, la concentration en glucose, la taille et la densité des cellules.Tout d'abord, le développement de colonies uniques de C. vulgaris a été étudié dans des conditions hétérotrophes. Sur la base du modèle biologique proposé pour la dynamique de croissance en hauteur et en rayon, nous avons conclu que les colonies se développaient à un taux constant dans la direction horizontale et à un taux décroissant dans la direction verticale. Ces tendances sont cohérentes avec les effets cumulés de la disponibilité du glucose et de l'oxygène. Une calotte sphérique décrit le mieux la forme des colonies pendant la période de croissance. L'interaction intraspécifique de C. vulgaris a été étudiée en faisant croître plusieurs colonies sur la même plaque à des distances de initiales différentes : 1,5 mm, 3 mm et 15 mm. Aucun effet significatif de la coalescence des colonies n'a été observé sur les taux de croissance en rayon et en hauteur.Ensuite, l'effet de la lumière a été testé de deux manières : présence de lumière durant toute la culture et exposition à la lumière après une première période hétérotrophe. La forme de la colonie est significativement affectée par le mode de culture : la colonie en croissance hétérotrophe garde une calotte sphérique, tandis que la colonie en croissance mixotrophe atteint une forme cylindrique, en raison d'une croissance radiale presque complètement arrêtée après quelques jours. Grâce au dispositif de mesure des gaz, les données brutes ont été analysées à l'aide d'une équation de bilan gazeux pour obtenir les termes source biologique d'O2 et de CO2. Le rapport de masse du gaz sur la masse sèche de la cellule a été déterminé pour les différentes conditions de croissance. Une synergie est mise en évidence entre la photosynthèse au sommet de la colonie et l’hétérotrophie à la base.L'interaction inter-espèces de C. vulgaris et S. cerevisiae a été étudiée à deux niveaux : au niveau des cellules dans la même colonie et entre colonies. Au niveau de la colonie, des colonies de C. vulgaris et de S. cerevisiae ont été inoculées avec deux distances de séparation initiales différentes (3 mm et 15 mm). Les colonies ont été observées en continu pendant un mois. Même si des recherches supplémentaires sont nécessaires, la croissance et l'interaction observées semblent s'expliquer principalement par le taux de croissance beaucoup plus élevé de la levure. Après avoir fusionné, les colonies de S. cerevisiae finissent par envelopper les colonies de C. vulgaris. Au niveau des cellules, les colonies mélangées de C. vulgaris et de S. cerevisiae ont été observées en 3D. En raison de leur croissance rapide, les cellules de S. cerevisiae finissent par dominer l'ensemble de la colonie, à l'exception de quelques cellules de C. vulgaris présentes au cœur de la colonie et sur le dessus. Les cellules de C. vulgaris cessent presque de croître lorsque les nutriments sont limités
Chlorella vulgaris (C. vulgaris) is a model organism that has high commercial potential in the food and energy field, with proved feasibility of cultures as biofilms and yeast/ microalgae co-culture for in situ CO2 mitigation in biotechnological processes. This PhD work focuses on immobilized colonies in pure or mixed cultures. It proposes a better understanding of the interactions within and between colonies, with the ultimate goal of understanding and optimizing co-cultures.To archive these goals, a comprehensive protocol and required innovative experimental devices were developed including inoculation techniques, immobilized culture devices with gas sensors, 3-D imaging using a structured-light microscope, image processing, calibrated gas balance equation and data analysis. Care was also taken regarding incubation conditions, determination of dry mass, glucose concentration, cell size and density.Firstly, the development of single C. vulgaris colonies under heterotrophic conditions was studied. Based on the biological model proposed for the growth dynamics in height and radius, we concluded that the colonies expanded at a constant rate in the horizontal direction and a decreasing rate in the vertical direction. The trends are consistent with the cumulative effects of glucose and oxygen availability. A spherical cap best describes the shape of the colonies during the growth period. The intraspecies interaction of C. vulgaris was investigated by growing several colonies on the same plate with different initial separation distances: 1.5 mm, 3mm, and 15 mm. No significant effects of colony merging were observed on the growth rates in radius and height.Then, the effect of light was tested in two ways: presence of light throughout the culture and exposition to light after a first, purely heterotrophic, period. The shape of colony is significantly affected by the cultivation mode: the heterotrophic growth colony keeps a spherical cap, while the mixotrophic growth colony reaches a cylindrical shape, due to a radial growth almost completely stopped after some days. Thanks to the gas measurement device, the raw data were analyzed using a gas balance equation to obtain the biological source terms of O2 and CO2. Gas yield (mass ratio of gas to dry mass of cell) are proposed for the different growth conditions. A synergy is highlighted between photosynthesis at the top of the colony and heterotrophy at the base.The interspecies interaction of C. vulgaris and S. cerevisiae were studied at two levels: cell-cell level within the same colony and colony-colony level. At the colony-colony level, colonies of C. vulgaris and S. cerevisiae were inoculated with two different initial separation distances (3 mm and 15 mm). Colonies were observed continuously for one month. Even though additional investigation is needed, the observed growth and interaction seems to be mostly explained by the much larger growth rate of yeast. After merging S. cerevisiae colonies eventually envelop C. vulgaris colonies. At the cell-cell level, C. vulgaris and S. cerevisiae intermixed colonies were observed in 3D. Due to its fast grow, S. cerevisiae cells eventually dominate the whole colony, at the exception of some C. vulgaris cells present in the core of the colony and on the top. C. vulgaris cells almost stop growing when the nutrients are limited

La variabilite de la taille et de la forme des semences de maïs a été caractérisée au sein d'une variété. Les effets de la taille de la semence et de sa forme plate ou ronde sur la croissance, le développement et le rendement en grain ont été étudiés au champ. Aucun effet de la forme de la semence n'a été mis en évidence. Le développement de la plante est faiblement influence par la taille de la semence. À la sortie de la phase d'hétérotrophie, la croissance est liée positivement au poids de la semence. Cet effet de la taille de la semence se maintient durant la croissance exponentielle, puis disparait en phase de montaison. Aucun effet de la taille de la semence n'a été mis en évidence sur le rendement en grain. En phase hétérotrophe, les écarts de croissance dus à la taille de la semence ne sont pas modifiés par un stress provoqué par des basses températures ou une contrainte hydrique

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ABSTRACT Microalgae include both algae Chlorophycea and cyanobacteria. These organisms have attracted interest due to its versatile metabolism, since some strains have, additionaly to photosynthetic metabolism, hability to grow in the dark by the consumption of simple organic molecules, such as glucose, acetate and glycerol. This hability suggests the application of these microorganisms in several areas, such as biological treatment of agroindustrial wastewater, aiming the removal of nutrients and organic matter. São Paulo/Brazil is one of the main producers of ethanol from sugarcane, generating a considerable amount of vinasse. Vinasse represents the main wastewater from de sugarcane agroindustry, being usually applied as fertigation in sugarcane crops. In this context, this study aimed to evaluate the heterotrophic growth of cyanobacterium Aphanothece microscopica Nägeli and the chlorophyceae Chorella vulgaris in synthetic culture media supplemented with organic carbon source and in vinasse, aiming to produce biomass and application it in wastewater treatment. Mixotrophic cultures were conducted to select the optimal concentration of organic substrate for the growth of microalgae, which were evaluated in heterotrophic cultures. Additionally, experiments have been conducted with pre-treated vinasse, evaluating the COD removal and the glucose and potassium incorporation. By the mixotrophic tests was selected the optimal concentrations of 25 and 12.5 g.L-1 glucose, 0.5 and 1.25 g.L-1 potassium acetate and 0.46 and 0.92 g.L-1 glycerol as ideal for the cultivation of Aphanothece microscopica Nägeli and Chlorella vulgaris, respectively, which reflected on the maximum specific growth rate between 0.0072 and 0.043 h-1. In the heterotrophic cultures the maximum specific growth rate was greater or equal to both microalgae in all carbon sources, with reduction between 30.4 and 90% of the initial concentrations of substrates. The experiments demonstrated possibility to use vinasse as culture media for both microalgae, especially Aphanothece microscopica Nägeli. In this conditions, results indicated the maximum removals of glucose (55.5%), COD (60.8%) and potassium (13%) by the cianobacterium, and 83.7% of glucose, 25% of COD and 13.8% of potassium by Chlorella vulgaris. The results suggest the heterotrophic metabolism for both microorganisms, pointing an interesting alternative to use of wastewater as culture media to produce high-value biomass from a low-cost way.
O termo microalga inclui tanto algas clorofíceas quanto cianobactérias. Recentemente estes micro-organismos têm despertado interesse devido ao seu metabolismo versátil, uma vez que algumas linhagens apresentam, além da fotossíntese como modelo de cultivo preferencial, a capacidade de desenvolverem-se no escuro a partir do consumo de moléculas orgânicas simples, tais como glicose, acetato e glicerol. Esta habilidade sugere a aplicação destes micro-organismos em diversas áreas, como o tratamento biológico de águas residuárias agroindustriais, visando a remoção de nutrientes e matéria orgânica. Apesar disso, são raros os trabalhos na literatura que avaliam o cultivo heterotrófico e o consumo ou incorporação de moléculas orgânicas por microalgas ou cianobactérias. O Estado de São Paulo responde por grande parte da produção nacional de etanol a partir da cana-de-açúcar, gerando uma quantidade considerável de vinhaça. A vinhaça é a principal água residuária do setor sucroenergético, sendo normalmente aplicada nos canaviais como fertirrigação. Neste contexto, o objetivo deste trabalho foi estender os estudos a respeito do cultivo mixotrófico e heterotrófico da cianofícea Aphanothece microscopica Nägeli e da clorofícea Chlorella vulgaris em meios com adição de fontes de carbono orgânico e em vinhaça, visando a produção de biomassa e aplicação no tratamento de águas residuárias. Ensaios mixotróficos foram conduzidos visando selecionar a concentração ideal de substrato orgânico para o cultivo das microalgas, as quais foram posteriormente avaliadas em sistemas heterotróficos. Paralelamente foram conduzidos ensaios com vinhaça, avaliando-se a remoção de DQO e incorporação de glicose e potássio pelas microalgas. Dos ensaios mixotróficos foram selecionadas as concentrações ótimas de 25 e 12,5 g.L-1 de glicose, 0,5 e 1,25 g.L-1 de acetato de potássio e 0,46 e 0,92 g.L-1 de glicerol como ideais para o cultivo de Aphanothece e Chlorella, respectivamente, refletindo em velocidades específicas de crescimento entre 0,0072 e 0,043 h-1. Nos ensaios heterotróficos verificaram-se velocidades específicas de crescimento iguais ou superiores para ambas as microalgas em todas as fontes de carbono avaliadas, com reduções entre 30,4 e 90% da concentração inicial dos substratos. Os ensaios com vinhaça demonstraram a possibilidade de utilização desta água residuária como meio de cultivo para ambas as microalgas, com alta conversão em biomassa pela Aphanothece. Nestas condições, verificaram-se remoções de 55,5% de glicose, 60,8% de DQO e 13% de potássio para a cianobactéria, e de 83,7% de glicose, 25% de DQO e 13,8% de potássio para Chlorella. Os resultados obtidos sugerem a presença do metabolismo heterotrófico nestes microorganismos e apontam uma alternativa interessante para o aproveitamento de águas residuárias, com a possibilidade de produzir biomassa de alto valor agregado a partir de um meio de baixo custo.

L’agriculture intensive est en pleine transition vers des pratiques agroécologiques qui s’appuient sur la biodiversité et les processus écologiques. Dans les agrosystèmes, la matière organique est un élément clé de la fertilité des sols et constitue une réserve importante de carbone. La fertilisation organique par les résidus de cultures est donc une pratique agricole qui permet d’améliorer le stock de matière organique. Les microorganismes du sol ont un rôle essentiel dans la dynamique du carbone et sont les principaux acteurs de sa minéralisation et de la mise à disposition des nutriments pour la plante. Ainsi, l’utilisation de biostimulant (BS) agricoles visant à améliorer cette fonction microbienne est proposée comme solution alternative pour améliorer indirectement la croissance des plantes tout en réduisant les intrants chimiques. A travers ces travaux de thèse il s’agissait i) d’identifier l’effet de biostimulants appliqués au sol sur les communautés microbiennes hétérotrophes, la minéralisation du carbone organique (Corg) et la libération des nutriments, ii) d’évaluer sa généricité en testant différentes conditions expérimentales et iii) d’identifier les filtres environnementaux qui contrôlent à la fois les communautés microbiennes et la fonction de minéralisation.Nous avons mis en évidence que la dynamique du Corg était différente en fonction des caractéristiques physico-chimiques et biologiques des sols. Nous avons montré que les plantes pouvaient elle aussi modifier la dynamique du Corg par le retour de leurs litières au sol et l’influence de leurs racines sur les communautés bactériennes et fongiques. Contrairement aux plantes, la quantité de Corg apportée par les BS étudiés étaient négligeables. Pourtant, nous avons évalué un effet parfois plus important que celui des plantes sur l’abondance, la diversité et la richesse des bactéries et des champignons. Parmi les deux BS étudiés nous avons montré que l’un améliorait la minéralisation du Corg en recrutant des bactéries et des champignons saprophytes indigènes du sol et que l’autre n’influençait pas la minéralisation du Corg mais activait des bactéries indigènes du sol promotrices de la croissance des plantes ainsi que des bactéries et champignons saprophytes. Notre étude souligne également l’importance de mettre en place une approche méthodologique normalisée et systémique qui intègre le suivi simultané de plusieurs descripteurs afin d’identifier les effets des BS
Modern agriculture is undergoing important changes toward agroecological practices that rely on biodiversity and ecological processes. In agrosystems, the organic matter is the key of the soil fertility and an important reserve of carbon. Organic fertilization by crop residues is therefore an agricultural practice that improve the organic matter content in soil. Soil microorganisms have an important role in the organic carbon (orgC) dynamic because they are key players of its mineralization and are involved in the nutrients recycling. Thus, the use of agricultural biostimulant (BS) intended to enhance this microbial function is proposed as an alternative solution to improve indirectly plant growth while reducing chemical inputs. This thesis aimed to i) identify the effect of soil biostimulant on heterotrophic microbial communities, the orgC mineralization and the nutrient releases, ii) evaluate its genericity by testing different experimental conditions and iii) identify the environmental filters that control both the microbial communities and the mineralization function. We showed that the orgC dynamic was different according to contrasted physico-chemical and biological characteristics of different soils. We showed that plants can also influence the orgC dynamic by returning litter to the soil and through its root effect on the bacterial and fungal communities. Unlike plants, the amount of orgC provided by the two tested BS was negligible. However, we evaluated the effect of one BS as at least similar or even higher than those of plant on active bacterial and fungal abundances, richness and diversity. Among the two BS tested we showed that one enhanced the orgC mineralization by recruiting indigenous soil bacterial and fungal decomposers and that the other did not affect the orgC mineralization but activated indigenous soil plant-growth-promoting bacteria as well as soil bacterial and fungal decomposers. Furthermore, our study call for new normative methodological and systemic approach by monitoring simultaneously several descriptors for advancing our knowledge on BS action on microbial soil functioning

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015
The increasing demand of corals, either for bioprospecting marine natural products for biomedical purposes, for the marine aquarium trade, or for utilization in coral reefs restoration efforts, has led to the need of cultivating these organisms. The production ex situ allows a better control over biomass production through the optimization of culture protocols. Therefore, it is important to understand the biology and physiology of cultivated organisms, in order to improve culture conditions, maximize growth and reduce production costs. These issues are highly relevant for the economic feasibility of coral aquaculture. Among a varied number of factors affecting coral growth, light (either the Photosynthetically Active Radiation – PAR, or the emitted spectrum) is one of the most important issues for symbiotic corals, due to their association with photosynthetic dinoflagellates of genus Symbiodinium (commonly termed as zooxanthellae). Additionally, the dynamics between autotrophy and heterotrophy also plays a key role in the success of coral growth. The present study was performed to evaluate the effect of: 1) light spectrum (white and blue light), 2) light PAR intensity (50 and 120 μmol quanta.m-2.s-1), 3) heterotrophic feeding (rotifers - Brachionus plicatilis (Müller, 1786)), in the physiology, photobiology and growth of fragments obtained from three mother colonies of the mixotrophic coral Sarcophyton cf. glaucum (Quoy & Gaimard, 1833), cultured ex situ in recirculated systems during 80 days. The supply of rotifers did not affect corals growth and promoted the accumulation of inorganic nutrients (nitrates and phosphates) in the culture water. The effect of light PAR intensities and spectrum as well as the interaction of these factors with heterotrophic feeding did not follow a similar pattern in coral fragments originating from different mother colonies. We concluded that the variability between colonies of the same species can play a key role in the response of corals to the studied parameters. Therefore, in a production scenario, mother colonies should be selected according their specific characteristics, to meet production objectives and culture conditions.
O aumento da procura de corais, tanto para bioprospecção de produtos naturais, como para o mercado da aquariofilia marinha, levou à necessidade de cultivo destes organismos. O cultivo de corais ex situ permite um maior controlo dos processos de produção, mas implica o desenvolvimento e otimização das infraestruturas e dos protocolos de cultivo. Para proporcionar condições ótimas de cultivo é essencial deter conhecimento da biologia e fisiologia dos organismos a cultivar, permitindo assim o seu desenvolvimento de uma forma rápida e saudável, a redução dos custos associados à produção, bem como a contribuição para a viabilidade económica da exploração. O cultivo de corais é afetado por diversos fatores, bióticos e abióticos. A luz (radiação fotossinteticamente ativa ou o espectro emitido) é um dos fatores abióticos mais importantes no cultivo ex situ de corais fotossintéticos, devido à relação ecológica de simbiose que mantêm com dinoflagelados do género Symbiodinium, vulgarmente designados por zooxantelas. Adicionalmente, o balanço entre autotrofia e heterotrofia desempenha um papel importante no sucesso do crescimento dos corais. O presente estudo foi desenvolvido com o objetivo de estudar o efeito de três fatores: 1) espectro de luz (luz branca e luz azul), 2) intensidade da radiação fotossintética ativa (50 e 120 μmol quanta.m-2.s-1) e 3) alimentação heterotrófica (fornecimento de rotíferos - Brachionus plicatilis (Müller, 1786)), na fisiologia, fotobiologia e crescimento do coral mole Sarcophyton cf. glaucum (Quoy & Gaimard, 1833), cultivado ex situ em sistemas recirculados, durante 80 dias. Os resultados obtidos revelaram que o fornecimento de rotíferos como alimento não beneficia diretamente a taxa de crescimento dos corais e promove um aumento de nutrientes inorgânicos na água de cultivo (nitratos e fosfatos). A resposta fisiológica ao espectro e intensidade de luz testados, bem como à interação destes fatores com a alimentação heterotrófica diferiu nos fragmentos de coral provenientes de diferentes colónias. Assim, conclui-se que a variabilidade entre colónias da mesma espécie influencia a resposta dos corais aos diferentes parâmetros estudados, pelo que num cenário de produção este aspeto deve ser acautelado através de uma seleção de colónias que reúnam determinadas características, em função dos objetivos de produção e das condições das instalações de cultivo.

A produção intensiva de produtos agrícolas, como os legumes e frutas, tem vindo a aumentar ano após ano e consequentemente, têm-se registado um aumento da produção de resíduos, derivados a transformação desses mesmos produtos. Muitas vezes este setor apresenta grandes dificuldades em tratar esses resíduos, muitos deles ricos em carbono orgânico, servindo como um substrato ideal para o crescimento de microalgas. Este trabalho de investigação teve como objetivo o estudo do crescimento de Chlorella protothecoides em quatro resíduos agroindustriais, o repiso de tomate, as borras de café, as borras de uva e o repiso de uva, que serviram como fonte de carbono orgânico. Face a estes resíduos, as borras de uva, foram o resíduo que apresentou melhores crescimentos apresentando uma taxa específica de crescimento 20% superior à taxa de crescimento em meio MCV. Realizaram-se quatro ensaios do tipo batch, onde se começou por utilizar meio MCV, como suplemento aos substratos de resíduo, terminando na utilização apenas dos resíduos como substrato para as culturas de crescimento. Verificou-se também que a microalga apresentou uma grande afinidade com o resíduo de repiso de tomate, registando-se na generalidade dos ensaios um pico de produção de biomassa deste resíduo. Neste ensaio ainda se verificou que a utilização de resíduos do setor da agroindústria, para o crescimento de microalgas, tem de ser cauteloso de forma a evitar que partículas sobrenadantes, provenientes dos resíduos, possam vir a influenciar na medição de parâmetros e ter um conhecimento claro da composição total destes resíduos de forma a detetar eventuais agentes inibidores do crescimento da C. protothecoides.
The intensive production of agricultural products, such as vegetables and fruits, increases year after year and, consequently, records an increase in waste production, derivative of the transformation of these same products. This sector often has great difficulties in treating these wastes, many of them rich in organic carbon, and because of that, it is an ideal substrate for microalgae growth. The objective of this research was to study the growth of Chlorella protothecoides in four agrowastes, which are tomato pomace, coffee grounds, grape pomace and grape grounds, which served as organic carbon source. Face these residues, the grape grounds, it was the residue wich shows a better microalgae growth, with a specific growth rate 20% higher than the growth rate in MCV medium. Four batch-type assays were performed, where began to use MCV media as a complement to waste substrates, ending only in the use of waste as a substrate for growing crops. It was also found that the microalga had a high affinity with tomato pomace, registering as a generality of tests a peak of biomass production in this agrowaste. In this essay, it was also achieved that the use of agrowastes for microalgae growth should be done carefully in order to prevent particules in supernatants from waste these wastes and having a clear knowledge of the total composition of these residues in order to detect any growth inhibitors of C. protothecoides it’s also important.

Dissertação de mestrado em Biotecnologia
The polyunsaturated essential fatty acids are divided into two relevant groups in human nutrition, omega-6 (n-6 PUFAs) and omega-3 (n-3 PUFAs) and carry on several important roles in the human body. However, several factors resulted in a consumption of n-6 PUFAs approximately 20 times greater than the consumption of n-3 PUFAs, showing a lack of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the dietary requirements. Both come from marine fish, such as salmon and mackerel, or from fish oils. As result of the increasing demand for food associated with a growing global population, these sources are not sufficient to answer the requirements for EPA and DHA, so it became necessary to explore alternative sources such as microalgae. The marine microalgae of the genera Nannochloropsis and Pavlova are important producers of EPA and DHA, since both exhibited the highest content of these n-3 PUFAS per dry weight biomass. Despite Nannochloropsis and Pavlova being currently used on the global food and feed market, the large-scale production of microalgae is not a widespread practice yet. The efficient use and wide implementation at an industrial scale requires improvements to reduce production costs. The main challenges to overcome are related with high nutrient supply and low production efficiency of biomass and high value-added metabolites. The potential of use agro-industrial by products and heterotrophic conditions as production strategies to reduce production costs and enhance microalgal biomass productivity of marine microalgae was evaluated with the following strains: Nannochloropsis gaditana, Nannochloropsis oceanica, Pavlova lutheri, Pavlova pinguis and Pavlova gyrans. The heterotrophic growth was evaluated with N. gaditana and N. oceanica. It was determined that both Nannochloropsis strains are not able to grow in heterotrophic conditions. P. pinguis and P. gyrans achieved similar or significant cell growth and lipid content in the medium composed of organic fertilizer than in synthetic control medium (f/2 medium). Cell growth and lipid content in the non-sterilized organic fertilizer medium were also found to be comparable to those in sterilized organic fertilizer media. P. gyrans cultured in organic fertilizer media suffered an interesting cell morphology variation, resulting in small, more elongated and motile cells. The use of synthetic aquaculture effluents as a culture medium to P. pinguis and P. gyrans cultivation, promoted an opposite trend, as lower cell growth was determined for both of these strains, suggesting the inhibitory impact of high nitrate concentrations (16 to 20 mM). The lipid content was unaltered. Overall, our results demonstrate that the replacement of synthetic culture medium by agroindustrial by-products is a viable approach to reduce production costs of Pavlova strains at industrial scale, while also contributing to a more sustainable economy and to produce organic microalgal biomass.
Os ácidos gordos polinsaturados estão divididos em dois grupos relevantes em termos nutricionais, omega-6 (n-6) e omega-3 (n-3), e desempenham variadas funções importantes no organismo humano. Contudo, diferentes fatores levaram a um consumo de ácidos gordos polinsaturados n-6 aproximadamente 20 vezes superior ao consumo de ácidos gordos polinsaturados n-3, ressaltando-se o baixo consumo de ácido eicosapentaenóico (EPA) e ácido docosahexaenóico (DHA), que advém de peixes de água salgada, como salmão e cavala, ou de óleos de peixe. Como resultado da crescente demanda por alimentos, associada a uma crescente população mundial, estas fontes de ácidos gordos não são suficientes para atender aos requisitos nutricionais de EPA e DHA. Tornou-se por isso necessário explorar fontes alternativas, como as microalgas. As microalgas marinhas dos géneros Nannochloropsis e Pavlova são importantes produtores de EPA e DHA, exibindo um elevado conteúdo de ácidos gordos n-3 por biomassa de peso seco. Apesar das espécies Nannochloropsis e Pavlova serem atualmente comercializadas a nível global, a produção em larga escala de microalgas ainda não é uma prática generalizada. Uma ampla implementação à escala industrial requer melhorias de forma a reduzir os custos de produção. Para atingir este objetivo, abordagens como (1) aumento da produtividade da biomassa por meio de culturas em condições de heterotrofia ou (2) substituição do meio de cultura sintético por subprodutos agroindustriais, podem ser implementadas. O potencial destas duas estratégias de produção foi avaliado com recurso às seguintes espécies de microalgas marinhas: Nannochloropsis gaditana, Nannochloropsis oceanica, Pavlova lutheri, Pavlova pinguis e Pavlova gyrans. Avaliou-se o crescimento heterotrófico das espécies N. gaditana e N. oceanica, e foi possível determinar que ambas as espécies não apresentam crescimento nestas condições. As microalgas P. pinguis e P. gyrans cultivadas em fertilizante orgânico obtiveram crescimentos celulares e teor lipídico semelhantes ou significativamente superiores às respetivas culturas controlo (meio de cultura f/2). Verificou-se também que o crescimento celular e o teor lipídico em meio de fertilizante orgânico não estéril são estatisticamente comparáveis aos obtidos com recurso a fertilizante orgânico previamente esterilizado. P. gyrans produzidas com fertilizante orgânico exibiram variação da morfologia das células, resultando em células mais pequenas, alongadas e com motilidade. A utilização de efluentes sintéticos de aquacultura como meios de cultura das espécies P. pinguis e P. gyrans promoveram um crescimento significativamente inferior ao obtido pelas respetivas culturas controlo (medi de cultura f/2), sugerindo um efeito inibitório de elevadas concentrações de nitrato (16 a 20 mM). O conteúdo lipídico manteve-se inalterado. De um modo geral, os resultados obtidos revelaram que a substituição de meios de cultura sintéticos por subprodutos agroindustriais é uma abordagem viável para reduzir os custos de produção de espécies de Pavlova à escala industrial, contribuindo simultaneamente para uma economia mais sustentável e obtenção de biomassa de microalgas certificada como orgânica.
Este trabalho foi realizado no âmbito do projeto ALGAVALOR - Microalgas: produção integrada e Valorização da biomassa e das suas diversas aplicações (POCI-01-0247-FEDER-035234), cofinanciado pelo Fundo Europeu de Desenvolvimento Regional (FEDER), Portugal 2020, através do Programa Operacional Competitividade e Internacionalização (COMPETE2020), do Programa Operacional Regional do Norte (Norte 2020), do Programa Operacional da Região Centro (Centro 2020), do Programa Operacional Regional de Lisboa (Lisboa 2020), do Programa Operacional Regional do Alentejo (Alentejo 2020) e do Programa Operacional do Algarve (CRESC ALGARVE 2020)