Nanoflagellates were found to be abundant in a coastal area of the northern Bothnian Sea. The maximum concentration of nanoflagellates, approximately 8000 cells ml-1, was observed in July, coinciding with a decrease in the abundance of cyanobacteria. Pigmented and non-pigmented nanoflagellates were approximately equally distributed throughout the year. Most of the identified genera are known as being phagotrophic, independent if autotrophic or not. A non-cyst-forming pigmented flagellate, Ochromonas sp., was isolated and nutritionally characterized. This chrysophycean flagellate was shown to be a mainly heterotrophic organism: Photosynthesis was too poor to support multiplication of the cells, whereas when feeding on bacteria, high growth rates were obtained. The biological function of the photosynthetic apparatus is suggested to be a survival mechanism during poor bacterial conditions. The flagellate grazed bacteria selectively, preferring cyanobacteria and large cells of heterotrophic bacteria, presumably depending on size-selective grazing. Despite higher growth rates of the bacteria in the sea during summer (July) than spring (May), heterotrophic bacteria in the sea was observed to be smaller in the summer. Nanoflagellates showed a maximum in July, and by selective grazing of large bacteria they might have caused the decrease in the average size of the bacteria and the decrease in the abundance of cyanobacteria. During the consumption of bacteria the flagellate was shown to remineralize nutrients at high rates and excrete dissolved free amino acids. Assuming the existence of a protozoan predator-prey chain of several trophic levels, it seems likely that a significant part of the nutrients fixed by primary producers is remineralized in the euphotic zone. Furthermore, data from this work indicate that flagellate activity may be a significant source of dissolved free amino acids, utilizable for the heterotrophic bacteria. / digitalisering@umu

Rampant increases in oil prices and detrimental effects of fossil fuels on the environment have been the main impetus for the development of environmentally friendly and sustainable energy sources. Amongst the many possibilities, microalgae have been proposed as a new alternative energy source to fossil fuels, as their growth is both sustainable and ecologically safe. By definition, microalgae are unicellular photosynthetic microorganisms containing chlorophyll a. These organisms are capable of producing large quantities of oils, surpassing that of traditional oil-seed crops, which can be transformed, through chemical processes, into biofuels such as biodiesel or bio-gasoline. Thus, recent research has gone into discovering high lipid producing algal strains, optimising growth media for increased lipid production and developing metabolic engineering to make microalgae a source of biofuel that is competitive to more traditional sources of biofuel and even to fossil fuel. In this context, the research reported here focused on using a mixotrophic growth mode as a way to increase lipid production for certain strains of microalgae. In addition, nitrogen starvation combined with mixotrophy was studied to analyse its effects on lipid production. Mixotrophy is the parallel usage of two trophic modes, in our case photoautotrophy and heterotrophy. Consequently, 12 algal strains were screened for mixotrophic growth, using glycerol as a carbon source. Glycerol is a waste product of the current biodiesel industry; it is a cheap and abundant carbon source present in many metabolic pathways. From this initial screening, several strains were chosen for subsequent experiments involving nitrogen starvation. Nitrogen starvation has been shown to induce lipid accumulation. The results obtained show that a mixotrophic growth mode, using glycerol as a carbon source, enhances lipid production for certain strains. Moreover, lipid enhancement was shown for nitrogen starvation combined with mixotrophic growth mode. This was dependant on time spent under nitrogen starvation and on initial concentrations of the nitrogen source. / L’augmentation effrénée des prix du pétrole et les effets néfastes des carburants fossiles sur l’environnement sont les raisons principales pour la recherche et le développement de nouvelles sources d’énergie durables et écologiques. Parmi de grands nombres de possibilités, les micro-algues sont proposées comme une source alternative d’énergie aux carburants fossiles, étant donné que leur croissance est durable et écologique. Les micro-algues sont des organismes unicellulaires et photosynthétiques détenant comme pigment essentiel la chlorophylle a. Ces organismes sont capables de produire de grandes quantités d’huile, parfois excédant celles des cultures agricoles traditionnellement utilisées pour les biocarburants. Ces huiles peuvent être transformées en biocarburants, tel que le biodiésel et le bio-essence, par certains procédés chimiques. La recherche actuelle est basée sur la découverte de souches d’algues capables de produire un haut rendement de lipides, l’optimisation de milieux de croissance pour accroitre la production lipidique et la manipulation génomique afin de créer des souches de micro-algues dont les rendements peuvent rivaliser avec l’agriculture tradionnelle et même les carburants fossiles. Dans ce contexte, notre recherche se concentre sur l’utilisation d’un mode de croissance mixotrophe afin d’induire une augmentation dans la production lipidique de certaines souches de micro-algues. De plus, des études sur la carence en azote combinée à la croissance mixotrophe ont été entreprises pour évaluer l’effet de ces paramètres sur la production lipidique. La mixotrophie est un mode de croissance qui utilise en parallèle deux modes trophiques différents, tel que l’hétérotrophie et l’autotrophie. De ce fait, 12 souches d’algues ont été examinées pour leur capacité à croitre dans un milieu mixotrophe. Le glycérol est un produit secondaire de l’industrie du biodiésel actuelle. Cette substance est à bas prix, abondante et peut être utilisé comme substrat dans plusieurs voies métaboliques. Du criblage initial, plusieurs souches ont été choisies pour des expériences subséquentes impliquant la carence en azote. La carence en azote à été démontrer comme un déclencheur de l’accumulation de lipide chez les micro-algues dans des recherches antérieures. Les résultats obtenus démontrent que la croissance mixotrophe permet d’augmenter la production de lipide chez certaines souches. De plus, la carence en azote combinée à la croissance mixotrophe a permis d’augmenter la production lipidique. Cependant, celle-ci dépendait du temps passer en carence et des concentrations initiales de source d’azote.

La production de microalgues en hétérotrophie présente plusieurs avantages pour la production de biocarburants par rapport à la production autotrophe, comme une productivité plus importante en termes de biomasse et de lipides. Cependant, le développement industriel de ce procédé est limité par les coûts de productions associés au substrat organique (i.e. glucose) et à ceux liés à la stérilisation des fermenteurs. Les effluents de fermentation sombre, composés principalement d’acétate et de butyrate, pourraient être utilisés comme milieux de culture peu onéreux pour la culture hétérotrophe ou mixotrophe de microalgues. Les objectifs de cette thèse étaient i) de mieux appréhender la croissance algale sur des mélanges variés d’acétate et de butyrate en fonction de la présence ou l’absence de lumière et de la température de croissance et ii) d’évaluer la faisabilité d’utiliser des effluents de fermentation non stérilisés pour soutenir la croissance de microalgues oléagineuses. Tout d’abord, un modèle basé sur des bilans de masse a été construit afin de caractériser la croissance hétérotrophe de Chlorella sorokiniana et Auxenochlorella protothecoides (taux de croissance et rendements) sur des mélanges d’acétate et de butyrate. Les résultats ont montré que le rapport acétate:butyrate et la concentration en butyrate étaient deux paramètres clés pour soutenir la croissance hétérotrophe. Puis, il a été démontré que la présence de lumière et l’utilisation d’une température suboptimale (30 °C) pour la croissance algale permettaient de réduire l’inhibition du butyrate en permettant une production de biomasse autotrophe ou en améliorant la croissance sur acétate. Enfin, il a été montré que les microalgues peuvent être compétitives sur l’acétate lors de la croissance sur des effluents bruts de fermentation sombre en présence de bactéries fermentaires, grâce à la croissance rapide des microalgues sur acétate (1.75 j-1) et à un changement drastique des conditions de culture peu favorables à la croissance des bactéries d’origine fermentaire. / Growing microalgae in heterotrophic mode present several advantages over autotrophic mode such as a higher productivity in terms of biomass and lipids for biofuels production. Nevertheless, this process is limited by the production cost associated with the organic substrate (i.e. glucose) and fermenters sterilization costs. Dark fermentation effluents, mainly composed of acetate and butyrate, could be used as a low-cost medium to grow microalgae heterotrophically or mixotrophically. The aims of this PhD were i) to optimize microalgae growth on various mixtures of fermentations metabolites using the presence or absence light and different cultivation temperatures and ii) to assess the feasibility of using unsterilized fermentation effluents. First, a model based on mass balance was built to characterize heterotrophic growth rates and yields when Chlorella sorokiniana and Auxenochlorella protothecoides were supplemented with different mixtures of acetate and butyrate. Results showed that the acetate:butyrate ratio and the butyrate concentration per se were two key parameters for promoting heterotrophic growth. Then, further studies showed that the presence of light and the use of suboptimal temperature (30 °C) could reduce the butyrate inhibition on growth by either triggering autotrophic production of biomass or enhancing growth on acetate. Finally, it was shown that microalgae could outcompete fermentation bacteria for acetate when growing on raw dark fermentation effluents, thanks to a fast algal growth on acetate (1.75 d-1) and a drastic change of culture conditions to the detrimental of bacterial growth.

<p>Temporal and spatial variations of pelagic microorganisms in the northern Baltic Sea were studied, as well as factors influencing their abundance and growth rates. Three main questions were asked 1) How does increased productivity influence the structure of the microbial food web? 2) Does predation limitation vary between trophic levels? 3) What is the relative importance of resource and predation limitation at different trophic levels?</p><p>A field study in the northern Baltic Sea showed that dominating protozoa, flagellates and ciliates, increased with increasing primary productivity from north to south. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. The relationship between plankton size structure and productivity was further studied in an experimental system. In agreement with present theories regarding nutrient status of pelagic food webs, increased productivity caused a lengthening of the food chain as well as a change in plankton size structure. While microplankton dominated in nutrient rich treatments pico- and nanoplankton dominated during nutrient poor treament. The flagellate community was dominated by a potentially mixotroph, <i>Chrysochromulina</i> sp., at low nutrient concentrations. To our knowledge this is the first experimental study showing that <i>Chrysochromulina</i> sp. in resemblance with other mixotrophs is favoured by nutrient poor conditions compared to strict autotrophs and heterotrophs.</p><p>During a stratified summer period autotrophic microorganisms in the northern Baltic Sea did not respond to removal of potential predators, indicating that they were primarily limited by inorganic nutrients. An exception was small eucaryotic picoplankton that showed a large response to predator removal. Among the heterotrophic microorganisms direct effect of predation seemed to increase from ciliates, heterotrophic bacteria, small heterotrophic flagellates, medium flagellates to large flagellates. No quick indirect effect was observed, but after four days trophic cascades were detected.</p><p>The relative importance of resource and predation limitation was studied among heterotrophic bacteria, flagellates and ciliates in the northern Baltic Sea. For all these groups, resource limitation seemed to prevail during the summer period. The results also indicated that the relative importance of predation increased with the productivity of the system. To our knowledge there are no earlier measurements on the relative importance of resource and predation limitation for micoorganisms in the pelagic environment.</p>

Temporal and spatial variations of pelagic microorganisms in the northern Baltic Sea were studied, as well as factors influencing their abundance and growth rates. Three main questions were asked 1) How does increased productivity influence the structure of the microbial food web? 2) Does predation limitation vary between trophic levels? 3) What is the relative importance of resource and predation limitation at different trophic levels? A field study in the northern Baltic Sea showed that dominating protozoa, flagellates and ciliates, increased with increasing primary productivity from north to south. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. The relationship between plankton size structure and productivity was further studied in an experimental system. In agreement with present theories regarding nutrient status of pelagic food webs, increased productivity caused a lengthening of the food chain as well as a change in plankton size structure. While microplankton dominated in nutrient rich treatments pico- and nanoplankton dominated during nutrient poor treament. The flagellate community was dominated by a potentially mixotroph, Chrysochromulina sp., at low nutrient concentrations. To our knowledge this is the first experimental study showing that Chrysochromulina sp. in resemblance with other mixotrophs is favoured by nutrient poor conditions compared to strict autotrophs and heterotrophs. During a stratified summer period autotrophic microorganisms in the northern Baltic Sea did not respond to removal of potential predators, indicating that they were primarily limited by inorganic nutrients. An exception was small eucaryotic picoplankton that showed a large response to predator removal. Among the heterotrophic microorganisms direct effect of predation seemed to increase from ciliates, heterotrophic bacteria, small heterotrophic flagellates, medium flagellates to large flagellates. No quick indirect effect was observed, but after four days trophic cascades were detected. The relative importance of resource and predation limitation was studied among heterotrophic bacteria, flagellates and ciliates in the northern Baltic Sea. For all these groups, resource limitation seemed to prevail during the summer period. The results also indicated that the relative importance of predation increased with the productivity of the system. To our knowledge there are no earlier measurements on the relative importance of resource and predation limitation for micoorganisms in the pelagic environment.

El comportamiento fagotrófico en especies fitoplanctónicas fue evaluado por medio de técnicas de análisis celular que permiten establecer patrones individuales de cada una de ellas. Primeramente, se calibró el contenido vacuolar como tasas de ingestión comparando la captación de microesferas fluorescentes de látex y el contenido de las vacuolas alimenticias por medio de la hibridación fluorescente in situ (CARD-FISH) en especies fitoplanctónicas bajo condiciones controladas de cultivo. Se obtuvo que el contenido de las vacuolas alimenticias caracterizado por la técnica del CARD-FISH refleja las tasas de ingestión obtenidas por la captura de microesferas al menos a escalas de horas. Así mismo, las tasas de digestión fueron diferentes entres protistas mixotróficos dependiendo de su tamaño celular, los individuos pequeños (<10 μm) digieren a una mayor velocidad que aquellos individuos de mayor tamaño. Posteriormente, se evaluaron los cambios a corta escala de tiempo (horas) de la actividad fagotrófica y los comportamientos selectivos en tres especies fitoplanctónicas (Cryptomonas ovata, Rhodomonas minuta y Dinobryon cylindricum) de un lago de alta montaña. Los resultados muestran diferencias temporales entre las especies, indicando cambios en las tasas de ingestión y digestión a lo largo del dia. Así mismo, se observó una clara preferencia por la ingestión de presas del dominio Archaea, la cual no puede ser explicada ni por la abundancia ni por el tamaño de las presas en el sistema. Finalmente, se comparo la actividad fagotrófica y la selección de presas en comunidades fitoplanctónicas de un conjunto de lagos de alta montaña para identificar generalidades de los patrones de ingestión observados. El patrón selectivo más general entre los diferentes protistas mixotróficos y en los diferentes sistemas estudiados es una preferencia sobre el grupo Actinobacteria, aunque esta preferencia puede ser muy variable. El tamaño celular de las presas es un parámetro importante en determinar las tasas de depredación selectiva que exhiben los mixótrofos sobre ellas. Así mismo, el tamaño celular de los protistas mixotróficos condiciona su actividad fagotrófica. Individuos con tamaños celulares grandes (>10 μm) presentan mayor contenido de presas en sus vacuolas, pero, debido a por su baja abundancia, el impacto sobre el bacterioplancton es bajo. Especies con tamaño pequeños (<10 μm) presentan un menor contenido, pero su alta abundancia y digestión más rápida hace que representen un mayor impacto sobre las comunidades bacterianas. La conclusión global de la tesis es que, incluso en condiciones óptimas de luz, la actividad fagotrófica de algunos grupos fitoplanctónicos típicos de los lagos de alta montaña (Chrysophyta y Cryptophyta) es muy importante, se han observado particularidades en el comportamiento y en la selección de presas y se ha evidenciado que el tamaño celular, tanto de los depredadores como de las presas, es el parámetro determinante para explicar las tasas de ingestión. / The feeding behaviour of phytoplankton species was evaluated through single cell analysis. Firstly, food vacuole content was calibrated as ingestion rate, comparing the intake of fluorescent microspheres and the prey vacuole content measured by Catalized Reported Deposition-Fluorescent in Situ Hybridization (CARD-FISH) on phytoplankton species in culture conditions. It has been observed that the food vacuole content properly reflects the ingestion rates at scales of hours. Likewise, digestion rates varied between species as a function of cell size: small individuals (<10μm) digest faster than bigger ones. Secondly, temporal changes of phagotrophic activity and selective behaviour were assessed in three phytoplanktonic species (Cryptomonas ovata, Rhodomonas minuta and Dinobryon cylindricum) in a high mountain lake. The results have shown that there are temporal differences between species, which indicates that changes in ingestion and digestion rates occurred along the day. A clear preference to ingest Archaea was observed in all three species. This preference could be explained neither by the abundance nor the size of the prey. Finally, the feeding behaviour of phytoplankton communities in a set of mountain lakes was compared, in order to identify general phagotrophic patterns. The most general selective behaviour in mixotrophic protists was a preference for Actinobacteria prey; however, this preference was highly variable and clearly related to the prey cell size. In addition, the cell size of mixotrophic protists determined their phagotrophic activity; small individuals (>10 μm), presented lower content of preys than bigger individuals, although, due to their higher abundance and faster digestion rates, they exerted a high grazing pressure on the bacterial communities. The main conclusion of this work is that, even under optimum light conditions, the phagotrophic activity in some typical phytoplankton groups of high mountain lakes (Chrysophyta and Cryptophyta), is significant; nevertheless, some particularities have been observed in the feeding behaviour of some species. Also, it has become clear that the size of both predator and prey are the most important parameters to explain ingestion rates.

La présente étude s’attache à étudier l’effet d’une source de carbone organique sélectionnée sur la croissance et la production de métabolites de la microalgue rouge Porphyridium marinum. Cette dernière s’est montrée incapable de se multiplier en hétérotrophie, en absence totale de lumière. Cependant, en condition mixotrophique la production de biomasse, lipides et de phycobiliprotéines par P. marinum a été améliorée en comparaison avec la condition autotrophique. Les teneurs en exopolysaccharides ont été presque similaires dans les deux conditions. Dans le but de valoriser ces métabolites, l’effet antioxydant, antibactérien, antibiofilm et anticancéreux ont été testés. L’exopolysaccharide de P. marinum ainsi que ses dérivés de faible poids moléculaire (EPS-2P et EPS-5P) ont tous présenté des activités antibactériennes et antibiofilm à différentes concentrations. Cependant l’EPS-2P et l’EPS-5P ont été jugés plus efficaces pour l’activité anticancéreuse contre les cellules de cancer de sein. D’autre part, la production du pigment majoritaire des microalgues rouges, la B-phycoerythrine (B-PE), a été optimisée moyennant des plans d’expériences adaptés. Une teneur de 40 mg/g MS a été obtenue en faisant varier la concentration en NaNO3, K2HPO4 et solution métallique ainsi que l’intensité lumineuse. Après purification, la molécule optimisée a montré une activité antioxydante en termes de piégeage des radicaux libres DPPH, chélation et réduction des ions de fer et inhibition de la décoloration du β-carotène. Nos données suggèrent alors que les métabolites produits par la microalgue rouge P. marinum peuvent être potentiellement utilisé dans plusieurs applications à savoir cosmétiques et pharmaceutiques. / The present study investigates the effect of a selected organic carbon source on the growth and production of metabolites of the red microalga Porphyridium marinum. The latter has been unable to multiply in heterotrophy, in total absence of light. However, in mixotrophic condition the productions of biomass, lipids and phycobiliproteins by P. marinum have been improved in comparison with the autotrophic condition. The contents of exopolysaccharide were almost similar under both conditions. In order to valorize these metabolites, the antioxidant, antibacterial, antibiofilm and anticancer effect were tested. The exopolysaccharide of P. marinum as well as its low molecular weight derivatives (EPS-2P and EPS-5P) have all exhibited antibacterial and antibiofilm activities at different concentrations. However, EPS-2P and EPS-5P were found to be more effective for anti-cancer activity against breast cancer cells. On the other hand, the production of the majority pigment of red microalgae, B-phycoerythrin (B-PE), has been optimized by means of adapted experimental plans. A content of 40 mg/g DW was obtained by varying the concentration of NaNO3, K2HPO4 and metal solution as well as the light intensity. After purification, the optimized molecule showed antioxidant activity in terms of free radical scavenging DPPH, chelation and reduction of iron ions and β-carotene bleachinginhibition. Our data suggest that the metabolites produced by the red microalga P. marinum may be potentially used in several applications namely cosmetic and pharmaceutical.

Odling av mikroalger för framställning av biogas är ett relativt outforskat område. Den forskning som hittills utförts har påvisat lovande resultat tack vare möjligheten att utnyttja resurser som idag går till spillo, eller till och med utgör miljöproblem; mikroalgerna kan rena både näringsrikt vatten (exv. avloppsvatten) och koldioxidutsläpp (rökgaser från industrin) då de tar upp föreningar innehållande kväve, fosfor och kol från dessa källor. Den producerade biogasen kan ersätta fossila bränslen. Dessutom skapas en rötrest som kan återföras till odlingsmarker vilket sluter näringskretsloppet. Mikroalgen Chlorella vulgaris undersöks gällande dess egenskaper i alg-biogasprocessen för att aktuella flöden ska kunna sammanlänkas genom industriell symbios. Mikroalger kan skapa unika möjligheter till kretsloppsanpassad energiproduktion bland annat tack vare att vissa av dem har potential att fungera både som växt och som djur. För att energiproduktionen ska kunna kretsloppsanpassas måste de olika systemen i alg-biogasprocessen lokaliseras strategiskt med avseende på dess flöden. / The area of microalgae cultivation for the production of biogas is quite uninvestigated. Research has shown promising results due to the possibility to make use of resources that are unused or even cause negative environmental impact: the microalgae can purify nutrient rich water (waste water) as well as exhausts rich in carbon dioxide (flue gas) as they take up compounds containing carbon, nitrogen and phosphorus from these sources. The produced biogas can replace fossil fuels. In addition, a digest is produced which can be returned to cropland which closes the nutrient loop. The microalgae Chlorella vulgaris is studied regarding its traits in the algae-to-biogas process in order to evaluate if the flows present can be interlinked through industrial symbiosis. Microalgae can create unique possibilities for loop adapted energy production partly thanks to their potential to function both as plant and as animal. For a loop adapted energy production the different systems in the algae-biogas process need to be located strategically with regards to the flows in the process. / The area of microalgae cultivation for the production of biogas is quite uninvestigated.Research has shown promising results due to the possibility to make use of resources that areunused or even cause negative environmental impact: the microalgae can purify nutrient rich water(waste water) as well as exhausts rich in carbon dioxide (flue gas) as they take up compoundscontaining carbon, nitrogen and phosphorus from these sources. The produced biogas can replacefossil fuels. In addition, a digest is produced which can be returned to cropland which closes thenutrient loop. The microalgae Chlorella vulgaris is studied regarding its traits in the algae-to-biogasprocess in order to evaluate if the flows present can be interlinked through industrial symbiosis.Microalgae can create unique possibilities for loop adapted energy production partly thanks to theirpotential to function both as plant and as animal. For a loop adapted energy production the differentsystems in the algae-biogas process need to be located strategically with regards to the flows in theprocess

Initially mycoheterotrophic plants have recently been declining in the wild, even without apparent causes. They are affected by a number of biotic and abiotic factors. The aim of this work is to investigate how selected factors, such as nitrate or symbiotic fungi, may influence their distribution in nature. In particular, the work applies in vitro experiments, molecular determination of fungal symbionts and stable isotope analyses. It describes both the effects of abiotic factor, specifically nitrate, and biotic interactions of initially mycoheterotrophic plants with fungal symbionts. The inhibition of germination by extremely low concentrations of nitrate in asymbiotic in vitro cultures was observed in several orchid species. The degree of sensitivity of each species to nitrate corresponds with the nitrate content of the soil and the nutrient availability requirements of the species according to Ellenberg indicator values. The inhibitory effect of nitrate on orchid germination was also observed in symbiotic in vitro cultures. Out of five tested fungal strains, only one Ceratobasidium was capable of eliminating the inhibitory effect of nitrate. Furthermore, the work reveals that green mixotrophic orchids use photosynthates to nourish the aboveground parts, whereas the belowground parts are...