Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.

Microalgae used in sewers to capture CO2 eventually turns into waste material. Through the use oftheir biomass, the waste algae can be given a new purpose. In this study attempts to extract starch or PHA from three different algae; Calothrix Scytonemicola, Scenedesmus Almeriensis and Neochloris Oleoabundans, were made. We also attempted to create a bio-based plastic material. Both Scenedesmus Almeriensis and Neochloris Oleoabundans are starch rich microalga. By washing with acetone, cryo grinding, use of ultrasonic homogenizer and dialysis, starch was likely extracted successfully. The extracted material and the plasticiser Carboxymethyl Cellulose (CMC) was used to cast plastic film. The cast film was very thin and brittle; perhaps by using different plasticisers or additives a more usable bio-based plastic material can be created. The PHA rich algae Calothrix Scytonemicola was used to extract PHA. The algae was washed with acetone, cryo grinded and then mixed with Sodium Hypochlorite(aq) and deionised water to extract the desired PHA. Due to a shortage of algae very small amounts of material could be extracted. Therefore, the casting of a plastic film was performed with commercial PH3B, which is a type of PHA. Three attempts were conducted. The first one with only chloroform, the second one with CMC and chloroform and the last one with Sucrose Octaacetate and chloroform. The film with Sucrose Octaacetate gave the best plastic material in regards to mechanical properties. / Mikroalger som används i kloaker för att binda CO2 blir till slut restavfall. Genom att använda dess biomassa kan restalgerna få ett nytt syfte. I denna studie utfördes extraktionsförsök av stärkelse samt PHA från tre olika alger, Calothrix Scytonemicola, Scenedesmus Almeriensis och Neochloris Oleoabundans. Ytterligare försök genomfördes för att försöka framställa ett biobaserat plastmaterial. Både Scenedesmus Almeriensis och Neochloris Oleoabundans är stärkelserika mikroalger. Genom att tvätta dem med aceton, kryomalning, användning av en ultrasonic homogenizer och dialys kunde stärkelse troligtvis extraheras. Det extraherade materialet blandades med karboxymetylcellulosa (CMC) för att skapa en plastfilm. Filmen blev väldigt tunn och spröd, således behövs antingen en annat mjukningsmedel eller tillägg av additiv för att skapa ett mer användningsbart biobaserat plastmaterial. Den PHA-rika algen Calothrix Scytonemicola användes vid extraktionen av PHA. Algerna tvättades med aceton och kryomaldes innan PHA förhoppningsvis extraheras med hjälp av natriumhypoklorit(aq) och avjonat vatten. På grund av en för liten mängd tillgänglig alg extraherades endast en liten mängd material. Det var därför inte möjligt att skapa en plastfilm av vårt extrakt utan istället användes kommersiell PH3B, som är en typ av PHA. Tre försök genomfördes, en med endast kloroform, en med CMC och kloroform och den sista med sucrose octaacetate och kloroform. Den sistnämnda filmen gav det bästa plastmaterialet med avseende på de mekaniska egenskaperna.

Mariculture has currently brought greatly valuable products in many fields simultaneously released a large amount of wastewater contributing to water pollutions on account of its organic and inorganic constituents. Nowadays, with the development of environmental engineering, more and more approaches, especially friendly-environmental and highly effective wastewater biological methods, are being applied to tackle pollutions and minimize adverse effects of treatments to reach the sustainable development. This report focuses on the study of proliferation combined with elimination of polluting substances of marine algae species Tetraselmis suiscica, Tetraselmis sp., Platymonas sp. in aquaculture wastewater sampled from Can Gio District, Ho Chi Minh City, Vietnam with levels of concentration during 14 days under normal marine algae culture conditions, and compared to that in Daigo’s IMK media. The results shown that, the algae species all grew rapidly simultaneously gave high nutrients removal yields (COD, N, P) and created a considerable amount of biomass within a short period of culture. Particularly, Platomonas sp. and Tetraselmis suiscica could proliferate aswell as give high treatment yields of organic substances (COD), PO43-, NO3-, NH4+ and Total Nitrogen in concentrated wastewater. To sum up, this study showed the potential of using microalgae to reduce COD, nitrogen and phosphorus in mariculture wastewater. / Ngành nuôi trồng thủy hải sản trong những nằm gần đây đã mang lại nhiều sản phẩm có giá trị trong nhiều lĩnh vực đồng thời thải ra một lượng lớn nước thải gây ô nhiễm nguồn nước bởi các thành phần vô cơ và hữu cơ có trong nước thải. Ngày nay, với sự phát triển của kỹ thuật môi trường, ngày càng nhiều cách tiếp cận, đặc biệt là các phương pháp sinh học hiệu quả cao và thân thiện với môi trường đang được ứng dụng để xử lý ô nhiễm và giảm thiểu hậu quả bất lợi sau xử lý, nhằm hướng tới sự phát triển bền vững. Bài báo cáo tập trung nghiên cứu khả năng sinh trưởng và phát triển của ba loài vi tảo biển Tetraselmis suiscica, Tetraselmis sp., Platymonas sp. kết hợp với việc loại bỏ các hợp chất gây ô nhiễm có trong nước thải từ ao nuôi tôm của huyện Cần Giờ, Việt Nam và so sánh đối chiếu với môi trường dinh dưỡng Daigo’s IMK trong 14 ngày nuôi trồng. Kết quả cho thấy những vi tảo sinh trưởng rất nhanh nhờ vào việc sử dụng các chất ô nhiễm trong nước thải, đặc biệt hiệu suất xử lý COD, PO43-, NO3-, NH4+ và nitơ tổng của Platomonas sp. và Tetraselmis suiscica rất cao thậm chí trong môi trường nước thải đậm đặc. Do đó, chúng tôi kết luận, vi tảo có tiềm năng rất lớn trong việc giảm nồng độ chất hữu cơ, phốt pho và nitơ trong nước thải nuôi trồng thủy hải sản.

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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

<p>Owing to the rise in fossil fuel prices, overall energy security concerns, and the current push towards green engineering; renewable and green fuels have seen an increase in interest in recent years. Two notable technologies in this green movement are the production of biodiesel from microalgae and the production of biogas from anaerobic digestion of waste biomass. Production of biodiesel from microalgae was studied extensively in the 80s through the early 90s and found to be economically infeasible given the technology of the time. However, recent literature has suggested that one possible method to improve the feasability of the process would be to combine it with an anaerobic digestor to provide nutrient and biomass recycling. For such a system, having accurate models of each process would be highly advantageous for optimal design and control. To this end this thesis moves towards this overall goal by examining and modelling the anaerobic digestion of the microalgae <em>Chlorella vulgaris</em>.</p> <p>Starting with a set of experimental data (anaerobic digestion of <em>Chlorella vulgaris</em>) provided by LBE-INRA, the minimum number of kinetic equations needed to predict the data are found using principal component style analysis. This number is found to be two to three reactions. Using this as a basis for model development, a mass balance model is developed around both two and three reaction cases. To date there is very little literature on the modelling of anaerobic digestion of microalgae and so kinetic laws are selected from the general anaerobic digestion models ``Anaerobic Digestor Model 1'' (ADM1) and ``Acidogenesis/Methanogenisis Model'' (AM2). Given that the kinetic laws were derived from general literature, model fitting is a must. To faciliate this process a novel systematic parameter identification procedure to locate identifiable parameter subsets within each model is presented. Applying this novel procedure to the provided data is seen to lead to promising identification results. Through these identification trials it is shown that the three reaction model best captures the dynamics of the system. This three reaction model serves as the basis for subsequent steady state optimality and sensitivity analysis. From these efforts it is shown that the predicted optimal curves match literature data very well but uncertainty in certain key parameter estimates lead to highly sensitive model predictions (and therefore low confidence). This leads to the conclusion that the developed model is capable of predicting the kinetics of <em>Chlorella</em> digestion but additional trials are needed to further refine the model fitting results.</p> <p>Coupling an anaerobic digester to a microalgal culture is currently considered one of the most promising avenues towards the production of renewable bioenergy, either in the form of biodiesel or biogas. Accurate mathematical models are crucial tools to assess the potential of such coupled biotechnological processes and help optimize their design, operation and control. This paper focuses on the compartment of anaerobic digestion of microalgae. Using experimental data for the anaerobic digestion of <em>Chlorella vulgaris</em>, a grey-box model is developed that allows good prediction capabilities and retains low complexity. The proposed methodology proceeds in two steps, namely a structural and a parametric identification steps. The fitted model is then used to conduct preliminary optimization for the production of biogas from <em>Chlorella vulgaris</em>. The results provide some insight into the potential for bioenergy production from the digestion of microalgae and, more generally, the coupled process.</p> / Master of Applied Science (MASc)

Bioprinting is considered a key technology for future space missions and is currently being established on the International Space Station (ISS). With the aim to perform bioink production as a critical and resource-consuming preparatory step already on Earth and transport a bioink cartridge “ready to use” to the ISS, the storability of bioinks is investigated. Hydrogel blends based on alginate and methylcellulose are laden with either green microalgae of the species Chlorella vulgaris or with different human cell lines including immortilized human mesenchymal stem cells, SaOS-2 and HepG2, as well as with primary human dental pulp stem cells. The bioinks are filled into printing cartridges and stored at 4°C for up to four weeks. Printability of the bioinks is maintained after storage. Viability and function of the cells embedded in constructs bioprinted from the stored bioinks are investigated during subsequent cultivation: The microalgae survive the storage period very well and show no loss of growth and functionality, however a significant decrease is visible for human cells, varying between the different cell types. The study demonstrates that storage of bioinks is in principle possible and is a promising starting point for future research, making complex printing processes more effective and reproducible.

Le traitement biologique des eaux usées urbaines et industrielles reste une activité ayant un impact négatif sur l’environnement et sur le changement climatique par l’émission des gaz à effet de serre (GES), notamment le CO2. Les changements innovants au niveau des procédés de traitement des eaux usées par l’intégration des flocs de microalgues-bactéries ont abouti à des procédés multitrophiques sans apport d’O2 et sans dégagement du CO2. Il s'agit d'une étude de faisabilité de ces flocs-MaB pour la photobioremédiation des polluants (organiques et minéraux) et pour la production de biomasse valorisable en bioénergie dans le cadre de l'économie circulaire. En présence de la lumière, les flocs-MaB ont été intégrés dans des photobioréacteurs à biomasse fixe afin d'assurer un traitement durable des eaux usées grâce aux échanges symbiotiques entre les micro-oragnismes en terme de nutriments et de gaz. L'encapsulation des flocs-MaB dans des billes de PVA-alginate a montré l'effet des conditions physico-chimiques et hydrodynamiques sur l'élimination des polluants et l'évolution multicellulaire des flocs au sein des réacteurs à multi-échelles. Par ailleurs, la biomasse multitrophique immobilisée sur des supports biodégradables d'olive (OPP) et sur des disques en PVC a assuré une meilleure performance des bioréacteurs à lit fluidisé et à disques rotatifs, respectivement, pour la bioremédiation des eaux usées. Les propriétés des supports (porosité, rugosité et structure) et les comportements hydrodynamiques ont été contrôlés pour favoriser l'attachement des biofilms multitrophiques. Le développement de biofilm montre l'effet des interactions multitrophiques entre les microalgues et les bactéries sur l'élimination des composés organiques (DCO) et nutriments (ammonium et phosphore). La biomasse des flocs-MaB a été récupérée et réutilisée pour le traitement du digestat liquide à l'issu du digesteur et pour améliorer la production de biométhane par une co-digestion anaérobie. Ce procédé multitrophique et intégré permet d'obtenir Zéro déchet à la sortie du processus / The biological treatment of urban and industrial wastewaters represents a process with a negative impact on the environment and on climate change through the emission of greenhouse gases (GHG), particularly CO2. In the presence of light, microalgae-bacteria flocs (MaB-flocs) have been integrated into photobioreactors with fixed biomass to ensure a sustainable wastewater treatment without O2 supply and CO2 release. The entrapment of flocs in PVA-alginate beads has shown the effect of physicochemical and hydrodynamic conditions on the elimination of pollutants and the multicellularity evolution within multi-scale bioreactors. In addition, the immobilization of biomass on biodegradable olive carriers and on PVC disks provided a better performance of fluidized bed and rotating discs bioreactors, respectively, for the bioremediation of wastewater. The properties of the supports (porosity, roughness, and structure) and the hydrodynamic behaviors have favored the attachment of multitrophic biofilms. Biofilm development shows the effect of multitrophic interactions between microalgae and bacteria on the organic compounds (COD) and nutrients (ammonium and phosphorus) removals. The MaB-flocs biomass was recovered and reused for the treatment of the digestate and to improve the production of biomethane by anaerobic co-digestion. This integrated multitrophic technology makes it possible to obtain zero wastes at the end of the process

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.