Dissertations / Theses on the topic 'Microalgues – Biotechnologie'

Ce travail visait à caractériser et produire deux enzymes originaux hydrolysant d'une part un polysaccharide pariétal d'algue rouge (le γ-carraghénane), et d'autre part un polysaccharide d'algue brune (le fucoïdane). Ces deux endo-hydrolases extracellulaires sont produites par deux bactéries marines saprophytes, P. Carrageenovora, et SW5. Suite à la purification des deux enzymes sauvages, leur[s] gène[s] ont été clonés puis séquencés. L'activité recombinante obtenue par surexpression dans E. Coli a validé les séquences obtenues. L'analyse de ces dernières montre l'architecture modulaire des deux protéines. Cependant, le domaine catalytique de la γ-carraghénase n'a pas pu être identifié. Il s'agit donc d'un membre orphelin des glycoside hydrolases, distinct des carraghénases préalablement caractérisées. A l'inverse, les similitudes partagées par la séquence de la fucoïdanase avec deux autres fucoïdanases, a permis de définir une nouvelle famille de glycoside hydrolases
This work aimed to characterize and produce two new biocatalysts which hydrolyze two polysaccharides extracted from the cell wall of red algae (γ-carrageenan) and brown algae (fucoidan). These extracellular endo-hydrolases are produced by two saprophytic marine bacteria, Pseudoalteromonas carrageenovora, (y-Proteobacteria), and SW5 (Bacteroidetes). Following the purification ofwild-type proteins, their genes were cloned and sequenced. The recombinant activity obtained by overexpression in E. Coli confirmed that the cloned sequences coded for corresponding enzymes. Sequence analysis showed that the enzymes have a modular structure. The catalytic domain of the γ-carrageenase was net identified. This enzyme is therefore different from previously described glycoside hydrolases, and aise distinct from previously known carrageenases. The fucoidanase sequence shares similarity with two other bacterial putative fucoïdanase and these three enzymes define a new glycosidase family

Nous avons optimisé la culture d'A. Platensis en bioréacteur. Nous avons caractérisé et mis au point un système de culture qui comporte un bioréacteur en sac plastique fixé à une table vibrante. Une étude comparative entre un système de référence de culture et le système vibré a démontré que la croissance d'A. Platensis est réalisable en milieu vibré. L'optimisation de la culture en milieu vibré a été initialement effectuée à 15000 Lux qui a permis d'obtenir une concentration cellulaire X de 1,0 g/L et une productivité (P) de 0,2 g/Lj. Ensuite, d'autres cultures ont été réalisées à 15000 Lux, puis exposées directement à 31700 Lux. Nous avons démontré qu'à cette dernière intensité, la croissance cellulaire est limitée en carbone. Les valeurs de X et P sont de 1,4 g/L et de 0,3 g/Lj respectivement. Dans ces conditions, la mort cellulaire apparaît à des valeurs de pH supérieures à 11. Pour satisfaire le besoin en carbone, nous avons effectué la culture en photoautotrophie et en mixotrophie avec un contrôle de pH par injection de CO2. Cela a permis de maximiser X et P. En photoautotrophie, X = 3,4 g/L et P = 0,49 g/Lj et en mixotrophie, X = 3,9 g/L et P = 0,57 g/Lj
In this work, we aim to develop and characterize a new adapted culture system for A. Platensis. It is based on surface aeration of a flexible pouch bioreactor fixed on a vibrating table. A comparative study between a culture reference system and the vibrated system shows that the growth of A. Platensis is suitable in vibrated culture. The vibrated culture has been firstly optimized by increasing the light at an intensity of 15000 Lux. In these conditions, X reached 1. 0 g/L and P was 0. 2 g/Ld. Furthermore, other cultures were placed at 15000 Lux and then exposed directly to 31700 Lux. It has been showed that at 31700 Lux, the cellular growth is limited by the carbon. The values X and P are respectively 1. 4 g/L and 0. 3 g/Ld. In these conditions, the cellular death appears at pH values higher than 11. To feed microalgae in carbon, we submitted the cellular growth to phototrophic and mixotrophic conditions under pH control using CO2 injection. It allowed to optimize X and P. In phototrophic conditions, X = 3. 4 g/L and P = 0. 49 g/Ld and in mixotrophic conditions, X = 3. 9 g/L and P = 0. 57 g/Ld

Le phytoplancton, qui regroupe un ensemble de microorganismes aquatiques photosynthétiques unicellulaires, est consommé depuis des millénaires par des civilisations d’Asie et d’Amérique du Sud. En effet, leur teneur parfois très importante en protéines ou en lipides en font une source intéressante d’acides gras essentiels, polyinsaturés (AGPI) notamment. Mes travaux ont été menés sur Tisochrysis lutea, une haptophyte déjà exploitée pour l’aquaculture, qui pourrait à terme se substituer aux huiles de poissons surexploités. En me basant sur le principe d’évolution de Darwin, j’ai soumis cette espèce à une pression de sélection d’intensité croissante, dans le but de faire émerger des individus ayant une teneur accrue en AGPI. La sélection a été réalisée dans des cultures en continue maintenues pendant 6 mois dans des photobioréacteurs automatisés (sélectiostats). Un consortium de douze souches a été soumis à des variations de température ou à des variations conjointes de lumière et de température en opposition de phase, d’amplitudes croissantes au cours du temps. Les résultats montrent que cette pression de sélection a permis un doublement de la teneur en lipides totaux (voire une multiplication par 5 pour certaines souches), sans perte de croissance ni de qualité nutritionnelle, les profils d’acides gras des lipides polaires (naturellement riches en AGPI), n’ayant pas été significativement modifiés. L’augmentation des lipides totaux se répercute donc directement sur celle de chaque acide gras. Une analyse génétique a mis en avant des mutations du génome de ces souches, signe que la population finale résulte d’un processus d’adaptation et non uniquement d’acclimatation
Phytoplankton gatherunicellular aquatic photosynthetic microorganisms. It has been used for millennia as a food source by civilisations from Asia and South America. On top of the high protein or lipid content they are also a promising polyunsaturated fatty acids (PUFAs) source. My work was carried out with Tisochrysis lutea, anhaptophyte cultivated for aquaculture which could in fine replace fish oils. Using Darwinian evolution principle, I imposed to this species an increasing selection pressure, with the objective to favour the individuals with an enhanced PUFAs content. Selection took place in continuous cultures maintained for 6 months in automated photobioreactors (sélectiostats). A consortium of twelve strains was exposed either to temperature oscillations, or to combined opposite light and temperature oscillations, with increasing amplitude. Results show that this selection pressure lead to a two-fold increase in total lipid content (five-fold increase for some strains), without any decrease in growth rate or nutritional quality, as the fatty acid profiles in polar lipids (naturally rich in PUFAs) were not significantly modified. The increase in total lipids is therefore associated to an increase in the cell content of each fatty acid. Genomic analyses revealed mutations in the genome, demonstrating that the final population results from an evolutionary process, and not simply from acclimation

Les hydrocarbures (HCs) sont prédominants dans notre économie actuelle (carburants, cosmétiques, chimie, etc.) mais sont quasi-exclusivement issus des ressources fossiles. Les problématiques de changement climatique et d’épuisement des ressources poussent les recherches vers l’étude et la domestication des voies de synthèse naturelles d’HCs. Lorsque j’ai commencé ma thèse, une enzyme de biosynthèse d’HC, l’acide gras photodécarboxylase (FAP) venait d’être découverte chez la microalgue Chlorella. J’ai d’abord caractérisé son homologue chez la microalgue modèle Chlamydomonas. Une étude phylogénétique de la famille des GMC oxidoréductases à laquelle appartient la FAP a permis d’identifier un large réservoir de de 200 FAPs putatives. La caractérisation biochimique de plusieurs d’entre elles a permis de montrer qu’une FAP fonctionnelle a été conservée lors des endosymbioses secondaires. Cela suggère que la FAP joue un rôle important chez les algues. Ce rôle a été étudié par une approche de génétique inverse chez Chlamydomonas. La caractérisation physiologique de mutants knockout a permis de démontrer le rôle de la FAP dans la synthèse d’HCs dans le chloroplaste et de mettre en évidence des modifications physiologiques transitoires. Des mécanismes de compensation à l’absence d’HCs restent donc à découvrir. Dans une dernière partie, j’ai développé une souche d’E. coli exprimant la FAP et une thioestérase. Cette souche produit en continu des HCs dans la phase gaz des cultures, ce qui permet une récolte facilitée du produit d’intérêt sous forme pure. Cette étude constitue une preuve de concept que la FAP pourrait être utilisée pour la production biosourcée d’HCs
Hydrocarbons (HCs) are predominant in our current economy (fuels, cosmetics, chemicals, etc.) but are almost exclusively derived from fossil resources. Climate change and resource depletion concerns are pushing research towards the study and domestication of natural HC synthesis pathways. When I started my thesis, a HC forming enzyme, the fatty acid photodecarboxylase (FAP) had just been discovered in the microalgae Chlorella. I first characterised its homolog in the model microalgae Chlamydomonas. A phylogenetic study of the GMC oxidoreductase family to which the FAP belongs has allowed identification of a large reservoir of 200 putative FAPs. Biochemical characterisation of several of them showed that a functional FAP was maintained during secondary endosymbiosis. This suggests that FAP plays an important role in algae. This role has been studied by a reverse genetic approach in Chlamydomonas. The physiological characterisation of knockout mutants demonstrated the role of FAP in the synthesis of HCs in chloroplasts as well as transient physiological changes. Mechanisms to compensate for the absence of HCs therefore remain to be discovered. In a last part, I developed a strain of E. coli expressing the FAP and a thioesterase. This strain continuously produces HCs in the gas phase of the cultures, which allows an easier harvesting of the product of interest in a pure form. This study is a proof of concept that FAP could be used for the biobased production of HCs

This master thesis focuses on reuse of waste water from industrial plant processing yellow phosphorus. Theoretical part summarizes physical and chemical properties of phosphorus, its transport in biosphere and its role in living organisms. Also the production process of the industrial plant is described. Waste water that contains waste byproducts is considered to be useful source of essential nutrients for economical large scale microalgae cultivation and development of biotechnological processes. In practical part, optimization of growth conditions for microalgae Chlorella pyrenoidosa Chick (IPPAS C-2) cultivation in medium based on wastewater from yellow phosphorus warehouse is presented.

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

Green microalgae are an important source of natural products such as β-carotene, and have recently become objects of intense study for producing biodiesel and valuable recombinant proteins. Application of chloroplast engineering in microalgae is limited by the availability of tools for genetically engineering the chloroplast of commercially important species. The phytoene desaturase gene of a previously isolated norflurazon tolerant mutant of Chlamydomonas reinhardtii was isolated and sequenced. A thymine to guanine transversion in exon 2 changes codon 131 resulting in a F131V mutation that is located in the NADP binding site domain on the primary structure. This mutation clusters with three conserved amino acids, whose substitution confers norflurazon tolerance in other species, in a pocket on a 3-D structure of the protein. The pocket identifies the target site of norflurazon. The side pocket is at the opening of a tunnel leading to the enzyme's NADP binding site. The mutant gene was cloned and used as marker for glass-bead mediated nuclear transformation of C. reinhardtii using direct selection with 5 μM norflurazon. Integration was by illegitimate recombination and transformants were able to grow in media containing 150 μM norflurazon. Transformants exhibited cross tolerance to fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wildtype. This allows mutant pds gene to act as a dual negative/positive selectable marker that is conditional on the herbicide used. The F131V mutation was introduced into a synthetic gene encoding a Dunaliella salina phytoene desaturase that contained codons used frequently in C. reinhardtii chloroplast genes. The 1.8 kbp CpPDS1 gene was assembled from 74 oligonucleotides by overlap PCR. The coding sequence was inserted into a Dunaliella tertiolecta chloroplast targeting vector that integrated the CpPDS1 sequence into the ycf3-trnL-rbcL region of the plastome. The resulting vector was transformed into D. salina and D. tertiolecta chloroplasts using particle bombardment with plasmid coated gold microprojectiles. Norflurazon tolerant colonies were isolated and the D. salina and D. tertiolecta clones were shown to contain a pds gene integrated in the plastome using PCR analyses. Transformation of the CpPDS1 gene into C. reinhardtii chloroplasts by rescue of an atpB mutation only gave rise to herbicide tolerant colonies if the presequence was removed. Industrial production of algae in large volumes is limited by the availability of light to drive algal growth. This problem was addressed by expressing fluorescent protein Katushka in the chloroplast of C. reinhardtii which converts yellow light to red light. The Katushka gene was transformed into chloroplasts using vector pB10, which was constructed to rescue a deletion in the chloroplast atpB gene in the mutant CC373 strain. The Katushka coding sequence was codon-optimised for expression in chloroplasts and expressed from three different promoter and 5' UTRs (atpA, atpB and psbD). C. reinhardtii wild type cells were able to grow under either blue or red LED lights but grew best when both were present. Wild type cell grew poorly in yellow LED lighting. Cells expressing Katushka in the chloroplast exhibited enhanced autotrophic growth in yellow light and under conditions where yellow light was present and red light was limiting. The improvement in growth was related to the levels of Katushka fluorescence detected in chloroplast transformants, which was highest for the atpA promoter and UTR.

La biomasse des microalgues tropicales a des vertus naturelles qui peuvent être utilisées dans une large gamme de bioproduits. Leur valorisation peut permettre une production durable et commercialement viable. En effet, les microalgues tropicales représentent une grande biodiversité et bénéficient de conditions environnementales favorables à une production à grande échelle. Dans ce contexte, cette thèse vise à étudier de nouvelles souches tropicales afin de connaître leur potentiel de valorisation dans le domaine des biotechnologies, et plus particulièrement sur trois aspects : énergie, nutraceutique et antifouling. Ce dernier domaine a été étudié dans le cadre du projet ANR-CD2I « BIOPAINTROP » dont l’objectif est la lutte écoresponsable contre le biofouling. Ces travaux s’orientent vers des applications biotechnologiques, mais aussi vers le développement des nouvelles méthodes de caractérisation de l’activité antifouling. Sur les 50 souches étudiées, certaines ont montré la production de métabolites d'intérêt tels que le glycosylglycerol, des lipides de qualité pour la nutraceutique et la production de biodiesel. La souche Amphidinium sp. (P-43) a conduit à un extrait méthanolique possédant une activité biologique significative. Son efficacité dans la lutte contre le biofilm a été démontrée. De plus, l'étude d'écotoxicologie réalisée laisse présager d'un faible impact environnemental
Biomass of tropical microalgae have natural virtues that can be used in a wide range of bioproducts. Their valuation can enable sustainable and commercially viable production. Indeed, tropical microalgae represent a large biodiversity and benefit from favourable environmental conditions for large scale production. In this context, this thesis aims to explore new tropical strains to determine their potential development in the field of biotechnology, particularly in three areas: energy, nutraceutical and antifouling. This field is studied in the project ANR-CD2I "BIOPAINTROP" whose objective is the eco-responsible fight against biofouling. These works target biotechnological applications, but also development of new methods to characterize antifouling activity.Of the 50 strains studied, some have shown interest in the production of metabolites such as glycosyl glycerol, quality nutraceutical and lipids for biodiesel production. The Amphidinium sp. (P-43) stain led to a methanol extract having biological activity of interest. Its efficiency against biofilm was demonstrated. Moreover, the ecotoxicology study has suggested a low environmental impact

Improving wastewater (WW) treatment process is a major issue in different parts of the world. For a developed country like the UK where eutrophication is a problem that causes environmental and economical losses, and for a developing country like Jordan that is considered one of the most water scarce countries in the world, it is crucially important to improve the quality of the WW for safe reuse. Applying microalgae for WW treatment and biomass production is an economical and environmentally friendly method. However, this method has some challenges that need to be addressed, such as microalgae species selection, harvesting of the microalgae and the large area footprint. In this research, the overall aim was to bioprospect for microalgae that are adapted to the wastewater treatment plants (WWTPs) and evaluate the obtained microalgae depending on specific criteria for a successful application in high rate algal ponds (HRAPs), then there were attempts to improve the phosphorus removal in microalgae to increase the efficiency of the treatment process and reduce the area footprint. Bioprospecting for indigenous microalgae to the WW took place from January to May 2014. Water samples were collected from wastewater treatment plants (WWTPs) in the UK and Jordan. Eight different microalgae isolates were identified from each country. The results showed the Chlorella, Scenedesmus and Desmodesmus are common genera between the two countries and dominated the obtained isolates from the UK and Jordan. The isolates were identified using 18S rDNA and ITS1 5.8S ITS2 DNA barcoding markers. It was difficult to identify some of the isolates at the species level, as the 18S rDNA is too conserved to differentiate between the closely related species and due to the relatively poor representation of algae in GenBank. Then the obtained microalgae isolates were evaluated by their growth, efficiency in removing nutrients (i.e. nitrogen and phosphorus) and the settleability of the microalgae by gravity. Depending on the results the microalgae species were ranked to come up with some promising candidates to be applied on large scale. From the UK, Avonmouth_12 (Av_12) and Avonmouth_10 (Av_10) and from Jordan, Jordan_18 (Jo_18) and Jordan_29 (Jo_29) were distinguished in their performance in the WW. Since phosphorus is a major cause of eutrophication in the fresh water and it is important to reduce the level of phosphorus in the released WW to the legally permitted limits, this research aimed to study the possibility of improving phosphorus removal by microalgae. Using Chlamydomonas reinhardtii as a model to optimise the protocol to be applied in parallel with Av_12, which is a promising microalga isolate that has been applied on large scale in HRAPs in Beckington WWTP, the strategy was to overexpress a Phosphorus Starvation Response (PSR1) gene. The transformation process was successful in C. reinhardtii but not in Av_12. There was an enhancement of the specific phosphate removal rate in the transformed microalgae isolate CC 1010_B2 and CC 1010_A6 in comparison to the wild type strain CC 1010.

Un nouveau procèdé pour la culture de microalgues a été conçu et développé. Ce procédé est basé sur l'amélioration simultanée du transfert de matière gaz-liquide et de la vitesse de photosynthèse dans un système venturi éclairé. Une étude de faisabilité a été menée sur le photobioréacteur avec plusieurs souches microalgales. Les concentrations en biomasse et les taux de croissance obtenus ont été importants et prometteurs. De plus, un modèle a été élaboré pour décrire le comportement d'une souche microalgale dans le procédé de culture. Les résultats du modèle ont été validés par rapport aux résultats expérimentaux, à l'acide de la simulation dynamique, effectuée sur le simulateur généralisé « SpeedUp ».

Les installations nucléaires sont génératrices de déchets liquides radioactifs qui doivent être traités avant leur rejet dans l'environnement. Le cobalt et l'argent radioactifs sont, après le tritium et le carbone 14, les principaux radionucléides rejetés par des réacteurs à eau pressurisée. Les traitements de décontamination d'effluents liquides actuellement mis en œuvre dans les installations nucléaires reposent sur des procédés physico-chimiques d'évaporation, de coagulation/floculation, de séparation de phase, de sorption et d'échange d'ions. Ces procédés conventionnels sont très efficaces mais présentent diverses limitations : ils ne retiennent pas ou peu le carbone 14 et, en situation accidentelle, ils ne sont pas faciles à mettre en œuvre pour traiter de grands volumes. Le développement de procédés innovants palliant à ces inconvénients est donc nécessaire. Les technologies de bio-remédiation pourraient être une alternative intéressante dans le secteur nucléaire mais très peu de procédés ont été proposés. Ce travail vise ainsi à développer une filière de traitement d'effluents nucléaires originale basée sur l'action d'une micro-algue photosynthétique, Coccomyxa actinabiotis, résistant aux rayonnements ionisants et accumulant les radionucléides et métaux toxiques. Les réflexions menées en collaboration avec les différents acteurs du projet ont permis d'établir un cahier des charges pour concevoir la filière de traitement et réaliser un pilote en tenant compte des contraintes associées au milieu nucléaire et à l'utilisation d'une matrice biologique. La filière est organisée en plusieurs opérations incluant d'une part la production et la récolte des micro-algues et d'autre part la décontamination de l'effluent. La faisabilité de chacune de ces opérations est tout d'abord étudiée à l'échelle laboratoire. Ainsi, les conditions opératoires et les outils de suivi, de contrôle, et d'optimisation relatifs aux étapes de (i) production de biomasse algale, (ii) séparation et/ou concentration de la biomasse par microfiltration et (iii) décontamination de l'effluent, en particulier l'élimination de l'argent 110m, du cobalt 60 et du carbone 14, sont recherchés. Le montage de la filière complète est ensuite proposé ; basée sur les résultats obtenus à l'échelle de laboratoire, la faisabilité de la bio-décontamination de radionucléides par la micro-algue à l'échelle pilote est également étudiée et démontrée. Ce travail de recherche permet donc d'envisager le développement d'une filière innovante de traitement des effluents liquides d'industries nucléaires et confirme la potentialité de certaines micro-algues à assurer l'élimination de polluants ciblés
Nuclear plants produce radioactive liquid wastes which are decontaminated before they are released. Radioactive cobalt and silver are the main radionuclides released by water pressurized reactor, after tritium and carbon 14. Liquid effluents are decontaminated by physic-chemical processes, such as evaporation, coagulation, sorption and ion exchange. These technologies are very efficient but cannot neutralize entirely the carbon-14 and, in the case of emergency situation, they are difficult to implement in order to decontaminate high amount of radioactive liquids. It is necessary to look for alternative decontamination methods. Bio-remediation technologies may constitute interesting alternatives in the nuclear field as well, but only a few bio-based technologies have been proposed. This work aims to develop a treatment unit based on the use of a photosynthetic micro-alga, extremely radio-tolerant and owning high capacity to concentrate radionuclides and toxic metals. The technical specification was draft to design the process and construct the pilot unit taking into account the constraints linked to the use of a biological matrix in a nuclear environment. The pilot-scale treatment unit, based on this micro-alga, includes different tasks to ensure the objectives of the process: algae have first to be produced in a growth medium and harvested before ensuring the treatment of the contaminated effluent. The feasibility of these operations is studied at laboratory scale. Operating conditions and monitoring and optimization tools for each step, (i) biomass production, (ii) biomass separation and concentration by microfiltration, (iii) effluent decontamination of silver-110m, cobalt-60, carbon-14, are sought. Based on the results obtained at laboratory scale, the feasibility of bio-decontamination of radionuclides by the micro-alga at pilot-scale is studied and demonstrated. Through this work, the development of an innovative process has to be considered for the decontamination of liquid effluents from the nuclear industry. This work confirms the high potential of algae to ensure the pollutants elimination

The study introduces and demonstrates the viability of the continuous flow reactor (CFR) system for the production of bio-crude oil (BCO) from wet microalgae. Preliminary experiments conducted in the CFR system in hot compressed water (HCW) were successful in converting wet microalgae into liquid BCO. However, the synthesis and aggregation of high boiling point (HBP) components of BCO and the accumulation of char in the tubular piping of CFR system were identified as the limiting factor to the viability of the system. The aggregation of HBP components and the accumulation of char result to system blockage which prevents the continuous flow of the liquefaction product mixture in the CFR system. Inhibiting the reactions leading to the formation of HBP components and char will improve the performance of the CFR system. Therefore, the study seeks to incorporate co-solvents in the liquefaction reaction media in an attempt to inhibit or minimize the prevalence of HBP components of BCO. As such, different co-solvents were screened for their influence on improving the quality of BCO with respect to its boiling point profile (BPP), initial and final boiling point, as well as the amount of char recovered from each experiment. Only one co-solvent was chosen for further exploration in the CFR system. Batch liquefaction reactor’s (BLR) made up of stainless steel were used to carry out the co-solvent screening experiments. These experiments were carried out at a constant temperature (280 °C), pressure (75 bar), and co-solvent concentration (10 wt.%), at varying residence times. Solvent extraction with dichloromethane (DCM) was performed on the liquefaction product mixture to separate the products, viz. BCO, char and water soluble components. The extracted BCO was analysed through simulated distillation (SimDis) to obtain the BPP. The BPP properties of the BCO samples, from different liquefaction media, and the amount of char recovered were highly influenced by the addition of a co-solvent. The final boiling point (FBP) of tetralin, heptane, and n-octanol BCO products were significantly reduced to below 500 °C for all tested residence times except at 20 minutes. The residence time also proved to be influential in the processing of wet microalgae. n-Octanol was selected as the optimal performing co-solvent and was used for the continuous liquefaction of wet microalgae in the CFR system. The CFR system was modified by adding a co-solvent feed line into the continuous system since n-octanol was insoluble in water. The n-octanol pump was set at different flow rates, 0.2, 0.3, and 0.4 g/min, which resulted in a concentration of about 10 wt.% in the reactor feed. The concentration of n-octanol had a significant influence on the BPP of BCO components. The FBP’s were reduced with an increase in n-octanol concentration. The initial boiling point (IBP) of n-octanol BCO was increased to just above 100 °C which was required for the stability of the BCO product. The components of BCO were identified by GCMS. n-Octanol also proved to affect the composition of the BCO with respect to its components. HCW BCO components were significantly different from those identified from n-octanol BCO. A second co-solvent (tetralin) was used to prove whether the difference on the components of BCO was affected by n-octanol. The results proved that indeed the addition of different solvents in liquefaction reaction media favours the formation of different components. The amount of char formed was also reduced when using a co-solvent. A decrease in the oxygen/nitrogen compounds was also observed in the presence of a co-solvent, thus improving BCO properties.

CAPES
As microalgas são organismos com grande versatilidade metabólica, capazes de utilizar fontes de carbono orgânicas, como a glicose, e inorgânicas, como o dióxido de carbono (CO2). Este pode ser fixado pelas microalgas a partir da atmosfera, de carbonatos solúveis na água ou de efluentes industriais quando acoplados em um sistema de tratamento. A pesquisa desenvolveu-se na Churrascaria Devon’s onde por um sistema biológico de tratamento com microalgas, as emissões gasosas e voláteis das grelhas assadoras são parcialmente tratadas. O objetivo deste trabalho foi otimizar a produção de biomassa de dois inóculos de microalgas (Desmodesmus subspicatus e mescla Devon’s), em sistemas abertos (caixas) e fechados de cultivo (Fotobioreatores Tipo Painel). Para monitorar o crescimento das microalgas o método de quantificação celular foi padronizado pela correlação de Pearson (R2) entre absorbância (682nm), clorofila-a (mg.m-3) e peso seco (g.L-1). Adicionalmente foram calculados os parâmetros, velocidade máxima específica de crescimento (μmax), produtividade máxima de biomassa (Pmax) e densidade celular máxima (DCM) dos cultivos. Usando as duas biomassas dos cultivos otimizados foram feitas as análises de composição bioquímica de lipídios, carboidratos e proteínas. Os resultados indicaram que no sistema aberto a fumaça foi a variável mais significativa na produção de biomassa, seguida da adição do meio CHU e do tipo de inóculo. D. subspicatus em regime fechado apresentou ao final de 21 dias de cultivo 1,63 g.L-1 de biomassa seca (733,32 mg.m-3 de Chl-a), valor cerca de seis vezes maior do que em cultivo aberto. O mesmo foi observado com a Mescla Devon’s que no mesmo período apresentou 1,27 g.L-1 de biomassa seca (690 mg.m-3 de Chl-a) no sistema fechado, valor cerca de três vezes maior do que em sistema aberto. Com os dados de alcalinidade e pH foi possível relacionar que o bicarbonato (HCO 3-) foi a forma de carbono inorgânica predominante ao longo do cultivo. A centrifugação mostrou ser um método eficiente de recuperação celular para ambas as microalgas, sendo que o tempo de centrifugação foi a variável mais significativa para D. subspicatus e a velocidade (rotações por minuto) para a mescla Devon’s. A quantificação bioquímica mostrou que a microalga D. subspicatus apresentou teor de lipídios de 16,77%, de carboidratos totais de 28,17% e de proteínas totais de 35,3%. Já a mescla Devon’s, apresentou teor de lipídios totais de 23,23%, de carboidratos de 27,60% e de proteínas totais de 31,9%, sobre o total de biomassa seca. Esses resultados ressaltam a possível utilização da biomassa de microalgas cultivadas em sistemas de tratamento de gases de efeito estufa, tanto no setor alimentar, cosmético como energético.
Microalgae are organisms with high metabolic versatility, capable to use organic carbon sources such as glucose or inorganic carbon sources, such as carbon dioxide (CO2). This can be fixed by microalgae from the atmosphere, from soluble carbonates dissolved on the water or from industrial effluents when coupled to treatment system. The research developed in a barbecue restaurant Churrascaria Devon’s where using a biological treatment system with microalgae, gaseous and volatile emissions are partially treated. The aim of this study was to optimize biomass production of microalgae from two different inoculums (Desmodesmus subspicatus and Devon's mix) in open (boxes) and closed (Panel Photobioreactor) cultivation systems. To monitor the growth of microalgae, the quantification cellular method was standardized by correlation (R2) between absorbance (682nm) Chlorophyll a (mg.m-3) and dry weight (g.L-1). Additionally were calculated some parameters as, maximum specific growth rate (μmax), maximum biomass productivity (Pmax) and maximum cellular density (MCD) of the cultures. Biochemical analyses for lipids, carbohydrates and proteins were performed in both biomasses from optimized cultivations. The results indicated that in the open system the smoke was the most significant variable in the production of biomass, followed by addition of medium CHU and inoculum type. The D. Subspicatus in a closed system shows the in the end of 21 days of cultivation was 1, 63 g.L-1 of dry biomass (733, 32 mg.m-3 Chl-a) six times above if compared with cultivation open systems. The same was observed with the Devon's mix, that during the same time shows 1, 27 g.L-1 of dry biomass (690 mg.m-3 Chl -a) in the closed system, a value three times larger than in an open system. With the alkalinity and pH values was possible to confirm that, the bicarbonate (HCO3-) was the predominant form of inorganic carbon during the cultivation. Centrifugation was an efficient method of cell recovery from both microalgae cultures; the centrifugation time was the most significant variable for S. Scenedesmus and the rotations per minute to Devon's mix. The biochemical quantification showed for the S. Scenedesmus microalgae 16,77 % of lipid content, 28,17% of total carbohydrates and total protein of 35,3 %. To the Devon’s mix, the total lipid content was 23,23 %, carbohydrates of 27,60% and total protein of 31,9 %, on total dry biomass. These results showed the possibility of using biomass of microalgae from treatment of greenhouse gases systems.in the food, cosmetic and energy industry.

Orientador: Telma Teixeira Franco, Eduardo Jacob Lopes
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: O objetivo da dissertação foi avaliar o potencial da produto de matérias graxas por microalgas em processos de sequestro de carbono e a qualidade do biodiesel produzido pela biomassa produzida. A biomassa de microalgas foi produzida em reatores de coluna de bolhas, a partir de meios de cultivo sintéticos, nas condições de concentração celular inicial de 100mg.L-1, reator isotémico operando em temperaturas de 35°C, intensidades luminosas de 11klux e aeração contínua de 1VVM com injeção de ar contaminado com 15% dióxido de carbono. Nesta dissertação de mestrado, as espécies de microalgas descritas pela literatura como produtoras de matérias graxas foram cultivadas em condições fotossintéticas e avaliadas pelo estudo da correlação entre a produto de biomassa e a produção de biodiesel. Nesta etapa, a produção mássica das gorduras e análise qualitativa dos lipídeos produzidos das diferentes espécies foram os fatores determinantes para escolha da microalga com potencial para produção de biodiesel. Uma vez definida a espécie com maior potencial de exploração (melhores parâmetros de conversão de biomassa em materiais graxos, rendimento e produtividade), a composição dos materiais graxos foi determinada para estudo da compatibilidade ao perfil desejado para produção de biodiesel, bem como a qualidade final do biodiesel produzido para cada espécie estudada
Abstract: The objective of the dissertation was to evaluate the potential of production of greases by microalgae in processes of carbon sequestration and the quality of biodiesel produced by the biomass produced. The microalgae biomass was produced in reactors, bubble column, based on synthetic culture media, under the initial cell concentration 100mg.L-1, isothermal reactor operating at temperatures of 35 °C, light intensities of 11klux and continuous aeration of 1VVM with injection of air contaminated with 15% carbon dioxide. In this master's thesis of microalgae species described in the literature as producers of raw grease were grown under photosynthetic and evaluated by the study of the correlation between biomass production and the production of biodiesel. In this step, the mass production of fats and qualitative analysis of lipids produced from different species were the determining factors for selection of microalgae with potential for biodiesel production. After determining the species with the highest potential for exploitation (best parameters for conversion of biomass materials in acids, yield and productivity), the composition of fatty material was determined to study the compatibility of the desired profile for biodiesel production and the final quality biodiesel produced for all species
Mestrado
Desenvolvimento de Processos Químicos
Mestre em Engenharia Química

The aim of the work was to examine microalgae growth and nutrient elimination in various diluted digestates in the first trial, then to study CO2 removal from a simulated biogas mixture by the same strain in the second trial. Scenedesmus SCCP K-1826 was cultivated in the digestate from Sundet biogas plant diluted 10, 20 and 30 times. The cultures were open-air with occasional CO2 injections to control pH. On day 15, the best growth was obtained in the 10 times diluted sample. COD, TN and TP removal efficiencies were similar in each bottle as the strain didn’t perform better in any specific dilution. The control proved that additional mechanisms other than photosynthesis contributed to digestate cleaning. Using the 10 times diluted sludge, Scenedesmus was grown in sealed flasks filled with simulated biogas (35.3 % CO2 + 32.3 % CH4 + 32.3 % N2). More algal biomass was produced in this batch culture. Nutrient removal efficiencies were close to the ones reached in the open-air flasks. After 10 days, 96 % of carbon dioxide was reduced. Methane content was depleted as well, possibly due to undesirable methane oxidizing bacteria which infiltrated the medium.

Due to the increasing climate change concerns, biofuels have attracted more attention in the energy field as potential alternative energy sources. Particularly, microalgal biofuel has stood out because of its higher fuel yield potential and lower water and land demand than terrestrial biomass. Because of its outstanding photosynthetic efficiency, the microalgal technology is also investigated by researchers around the world as a potential biological solution for carbon capturing in the industrial sector. To explore the prospects of microalgal technology in a local context, this research lays it focus on investigating the potentiality of microalgal biofuel in the cement industry on Gotland, which is the largest emitter of greenhouse gases on the island. For this purpose, the thesis implements a series of estimations based on the emission data of Cementa AB, Slite, a picture of the potential production of algal biomass and biofuel was created, followed by comparisons to the energy situation on Gotland. While practical data of the selected microalgae species are presented, the results indicate a high potential of microalgae in the production of algal biofuel and the possibility for algal biofuel to power the industrial sector of Gotland, or even the island entirely. Although the estimations are made based on an assumption where all controlling parameters are assumed to be perfectly manipulated, the results still indicate the significance of microalgal technologies in the near-future bioeconomy and global energy system.

Spirulina (Arthrospira) platensis (Cyanophyceae) é uma cianobactéria de grande potencial econômico para a produção de proteínas, principalmente. A utilização simultânea de duas fontes de nitrogênio no cultivo deste microrganismo pode aumentar a quantidade final de biomassa produzida e possibilitar resultados interessantes do ponto de vista de processo de produção. Esta pesquisa teve como principal objetivo verificar o efeito da adição de nitrato de potássio (KNO3) em cultivos de S. platensis realizados com cloreto de amônio (NH4Cl). Através da utilização de planejamento fatorial completo (22 mais configuração estrela), foram verificadas proporções de concentrações iniciais de KNO3 e totais de NH4Cl que variaram de 5,10 a 24,9 mmol · L-1. Foram realizados oito cultivos experimentais e seis cultivos de ponto central, com a utilização de processo descontínuo alimentado e vazões exponencialmente crescentes de adição do sal de amônio. As variáveis independentes (quantidade das fontes de nitrogênio) foram avaliadas simultaneamente através de metodologia de superfície de resposta (regressão multivariável) quanto a sua influência nas variáveis dependentes: concentração celular máxima (Xm), expressa em massa seca por litro de meio, produtividade em células (PX) e fator de conversão de nitrogênio em células (YX/N). A otimização dos cultivos em relação à concentração celular resultou nos seguintes valores: KNO3 = 15,4 mmol · L-1 e NH4Cl = 14,1 mmol · L-1, com Xm esperado de 4450 mg · L-1. O maior valor de concentração celular obtido sob essas condições foi de 4327 mg · L-1, valor este equivalente ao obtido em cultivo de referência para KNO3 (KNO3 = 25,4 mmol · L-1; 4318 mg · L-1) e superior ao obtido em cultivo de referência para NH4Cl (NH4Cl = 21,5 mmol · L-1; 2125 mg · L-1). Concluiu-se que a adição de KNO3 em cultivos com NH4Cl contribuiu para que não houvesse deficiência de nitrogênio no decorrer dos cultivos, proporcionando maiores valores finais de biomassa, com maior conteúdo de clorofila, lipídios e proteínas. A utilização de NH4Cl permitiu a redução dos custos com o meio de cultura, tornando cultivos com associação de tais fontes de nitrogênio uma alternativa vantajosa para a produção desta cianobactéria.
Spirulina (Arthrospira) platensis (Cyanophyceae) is a cyanobacterium which has a great economic potential to protein production, particularly. The simultaneous use of two nitrogen sources can increase the final amount of produced biomass and provide interesting results from the production process point of view. This research had as main goal to evaluate the effect of potassium nitrate (KNO3) addition to S. platensis cultures performed with ammonium chloride (NH4Cl). Through the use of full factorial design (22 plus star configuration), proportions of initial KNO3 and total NH4Cl concentrations, which varied from 5.10 to 24.9 mmol · L-1, were validated. Eight experimental cultures and six central point cultures were carried out using fed-batch process and exponentially-increasing ammonium salt feeding rates. The independent variables (nitrogen sources amount) were simultaneously assessed by response surface methodology (multivariable regression) as to their influence on dependent variables: maximum cell concentration (Xm), expressed in dry mass per liter of medium, cell productivity (PX) and conversion yield of nitrogen to biomass (YX/N). The cultures optimization regarding cell concentration resulted in the following values: KNO3 = 15.4 mmol · L-1 and NH4Cl = 14.1 mmol · L-1, with an expected Xm of 4450 mg · L-1. The highest cell concentration value obtained under these conditions was 4327 mg · L-1, which is comparable to the cell concentration obtained in KNO3 reference culture (KNO3 = 25.4 mmol · L-1; 4318 mg · L-1) and superior to the one obtained in NH4Cl reference culture (NH4Cl = 21.5 mmol · L-1; 2125 mg · L-1). It was drawn that the addition of KNO3 to NH4Cl cultures contributed to avoid nitrogen deficiency during cultivation, providing higher final biomass values with higher chlorophyll, lipid and protein contents. The utilization of NH4Cl allowed the reduction of culture medium costs, turning cultures developed with such nitrogen sources association into an advantageous alternative to the production of this cyanobacterium.

High rate algae oxidation ponds (HRAOP) for domestic wastewater treatment generate biosolids that are predominantly microalgae. Consequently, HRAOP biosolids are enriched with minerals, amino acids, nutrients and possibly contain plant growth regulator (PGR)-like substances, which makes HRAOP biosolids attractive as fertiliser or PGR. This study investigated HRAOP biosolids as a starting material for a natural, cost-effective and readily-available eco-friendly organic fertiliser and/or PGRs. Various HRAOP extract formulations were prepared and their effect on plant growth and development was evaluated using selected bioassays. Initial screening included assessing the effect on change in specific leaf area, radish cotyledon expansion as an indicator of PGR-like activity, and seed germination index (GI). More detailed studies on fertiliser efficacy and PGR-like activity utilised bean (Phaseolus vulgaris) and tomato (Solanum lycopersicum) plants. Combined effects of sonicated (S) and 40% v/v methanol (M) extract (5:1 SM) had impressive plant responses, comparable to Hoagland solution (HS). Other potentially fertiliser formulations included 0.5% M, 1% M, 2.5% S and 5% S formulations. The 5:1 SM and 5% S showed greater PGR-like activity, promoting cotyledon expansion by 459 ± 0.02% and 362 ± 0.01%, respectively. GI data showed that none of the formulations negatively impacted germination. Further investigation showed that the 5% S formulation increased leaf length, width and area by 6.69 ± 0.24, 6.21 ± 0.2 mm and 41.55 ± 0.2 mm². All formulated fertiliser extracts had no adverse effect on chlorophyll content and plant nutrient balance as indicated by C:N (8-10:1) ratio. In addition, plants appeared to actively mobilise nutrients to regions where needed as evidenced by a shift in shoot: root ratio depending on C, N and water availability. Furthermore, 5% S caused a 75% increase in tomato productivity and had no effect on bean productivity. Whereas, 5:1 SM and 1% M formulation improved bean pod production by 33.3% and 11%, respectively but did not affect tomato production. Harvest index (HI) however indicated a 3% reduction in tomato productivity with 5:1 SM and little or no enhancement in bean productivity with both 5:1 SM and 5% S treatments. Bean plants treated with 5:1 SM and 5% S produced larger fruits, which could be an indication of the presence of a PGR effect. Overall, HRAOP biosolids extracts prepared and investigated in this study demonstrated both fertiliser characteristics and PGR-like activity with performances comparable and in some cases exceeding that of commercial products. However additional research is needed to confirm presence of PGR-like activities and fertiliser efficacy.

Microalgae have a variety of commercial applications, the oldest of which include utilisation as a food source and for use in wastewater treatment. These applications, however, are seldom combined due to toxicity concerns, for ethical reasons, and generally the requirement for cultivation of a single algae species for use as a feed supplement. These problems might be negated if a “safer” wastewater such as that from agricultural and/or commercial food production facilities were to be utilised and if a stable algae population can be maintained. In this investigation preliminary studies were carried out using an Integrated Algae Pond System (IAPS) for domestic wastewater treatment to determine the species composition in the associated High Rate Algae Ponds (HRAPs). The effect of different modes of operation, continuous versus batch, on nutrient removal, productivity and species composition was also investigated. Furthermore, indigenous species in the HRAP were isolated and molecularly identified as, Chlorella, Micractinium, Scenedesmus and Pediastrum. Additionally, the effect of the nor amino acid, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA) and its Cu-chelated derivative, on the growth and biochemical composition of Chlorella, Micractinium, Scenedesmus, Pediastrum and Spirulina was investigated. Species composition in the HRAP was stable under continuous operation with Micractinium dominating > 90% of the algae population. Under batch operation the population dynamic shifted; Chlorella outcompeted Micractinium possibly due to nutrient depletion and selective grazing pressures caused by proliferation of Daphnia. Higher species diversity was observed during batch mode as slower growing algae were able to establish in the HRAP. Nutrient removal efficiency and biomass productivity was higher in continuous mode, however lower nutrient levels were obtained in batch operation. HMTBA did not significantly affect growth rate, however treatment with 10 mg.L-1 resulted in slightly increased growth rate in Micractinium and increased final biomass concentrations in Chlorella, Micractinium and Spirulina (although this was not statistically significant for Micractinium and Spirulina), which are known mixotrophic species. Algae treated with Cu-HMTBA, showed reduced final biomass concentration with 10 mg.L-1, caused by Cu toxicity. Biochemical composition of the algae was species-specific and differed through the growth cycle, with high protein observed during early growth and high carbohydrate during late growth/early stationary phase. Additionally, 0.1 mg.L-1 HMTBA and Cu-HMTBA significantly reduced protein content in Chlorella, Micractinium, Scenedesmus and Pediastrum. In conclusion, operation of the HRAP in continuous culture provided suitable wastewater treatment with high productivity of an ideal species, Micractinium, for use in animal feed supplementation. This species had 40% protein content during growth (higher than the other species tested) and dominated the HRAP at > 90% of the algae population during continuous mode. Addition of HMTBA (> 1 mg.L-1) to algae cultivation systems and those treating wastewater, has the potential to improve productivity and the value of the biomass by enhancing protein content. Overall, the co-utilisation of microalgae for wastewater treatment and the generation of a biomass rich in protein, for incorporation into formulated animal feed supplements, represents a closed ecosystem which conserves nutrients and regenerates a most valuable resource, water.

Microalgae can be grown on municipal wastewater media to both treat the wastewater and produce feedstock for algae biofuel production. However the reliability of treatment must be demonstrated, as well as high areal algae productivity on recycled wastewater media and efficient sedimentation harvesting. This processes was studied at pilot scale in the present research. A pilot facility was operated with nine CO2-supplemented raceway ponds, each with a 33-m2 surface area and a 0.3-m depth, continuously from March 6, 2013 through September 24, 2014. The ponds were operated as three sets of triplicates with two sets continuously fed primary-clarified municipal wastewater at either a 2-day or 3-day hydraulic residence time (HRT), and one set fed the clarified effluent of the 3-day pond set. This second pond-in-series was operated with a 3-day HRT. Areal biomass productivity is reported as gross and net, the former based only on biomass in the pond effluents and the latter subtracting the volatile suspended solids in the influent from those in the effluent. An estimate was also made of autotrophic biomass productivity, as differentiated from heterotrophic growth. Over a year, net productivity averaged 83 metric tons per hectare per year (MT/ha-yr) for the 2-day HRT ponds, 52 MT/ha-yr for the 3-day HRT ponds, and 44 MT/ha-yr for the 3-day HRT ponds receiving clarified effluent of the first set of 3-day HRT ponds (i.e., recycled water). The lower net productivity of the pond receiving water recycling was attributed to two factors. First, the relatively high influent suspended solids concentrations were subtracted from the effluent suspended solids concentrations before net productivity was calculated. Second, the recycled water contained less soluble organic matter than the primary-clarified wastewater leading to less heterotrophic biomass production. The accumulation of inhibitory allelochemicals is a possible third cause of lower productivity , but no specific information was collected on allelopathy. Algae were harvested from pond effluent by sedimentation, with harvest efficiency most affected by the extent of natural bioflocculation occurring in the ponds. Some forms of bioflocculation are thought to be mediated by bacteria, which often make-up a substantial fraction of the settled flocs. Pond samples settled in 1-L Imhoff cones averaged/L total suspended solids after 24 hours of settling; but all ponds fell short of meeting an averaged/L total suspended solids after a 2 hour interval which would be ideally achieved for wastewater effluent. No relationship was seen between settling performance and the bacterial content of flocs. Soluble carbonaceous biochemical oxygen demand (scBOD5) removal by the raceway ponds was sufficient to meet wastewater treatment requirements year around. Influent scBOD5 concentrations averaged 83 mg/L, and the effluent averaged 5.1 mg/L and 4.2 mg/L for the 2-day and 3-day HRT pond sets, respectively. The variable with the greatest influence on productivity in all pond sets, and settling performance in the recycled water pond set, was season (i.e., co-correlated variables of solar insolation and pond temperature). Neither productivity nor settling appeared to be related to prominent algae genera or prevalence of grazers. The high net productivity achieved with a growth medium of primary clarifier effluent and the generally high settleability of algal-bacterial flocs indicate a good potential for algae wastewater treatment and biofuel production. However, the settling of algae grown on recycled water needs improvement to achieve the full potential of wastewater-grown algae biofuel production.

Des biocarburants alternatifs, utilisant des microsystèmes microbiens, sont actuellement étudiés dans le but de limiter la consommation non raisonnée de ressources énergétiques et le rejet de gaz à effet de serre, qui modifient le climat. Dans cette thèse, nous avons considéré des bioréacteurs à base de microalgues oléagineuses, et des écosystèmes bactériens anaérobies qui décomposent des déchets et produisent du méthane . Ces travaux avaient pour objectif de mieux comprendre ces procédés et d'en améliorer les performances. Nous avons tout d'abord modélisé et étudié des cultures de microalgues en photobioréacteurs, dans lesquels les pigments algaux induisent une forte atténuation lumineuse. Pour les écosystèmes bactériens, nous avons utilisé un modèle précédemment développé. A l'aide de ces modèles et de leurs analyse mathématique rigoureuse, nous avons proposé des stratégies pour optimiser leur productivité. Ensuite, l'étude de la sélection naturelle entre plusieurs espèces de microorganismes dans ces deux écosystèmes a permis de prédire quelles espèces remportent la compétition. Et finalement nous avons montré comment il est possible, dans chaque écosystème, de contrôler la compétition pour diriger la sélection naturelle de façon à avantager des espèces ayant des caractéristiques permettant une performance accrue.

Les microalgues sont l’une des ressources renouvelables les plus prometteuses pouvant constituer une alimentation durable future. Grâce à leur diversité de métabolisme, ces microorganismes sont capables de synthétiser une vaste gamme de composés d’intérêt à haute valeur nutritionnelle. Cependant, leur consommation reste limitée du fait de leurs caractéristiques organoleptiques intrinsèques peu attrayantes. Afin de répondre à cette problématique et de lever les verrous, les travaux de cette thèse ont porté sur la mise au point d’un procédé de production d’ingrédient alimentaire à partir de spiruline.Une méthode verte et innovante faisant intervenir la technologie ultrasonore pour l'extraction de protéines à partir d'Arthrospira platensis a été proposée dans une première partie. Il s’agit de la manothermosonication (MTS). Le recours à un plan d’expérience a permis d’optimiser les paramètres d’extraction ; et une modélisation mathématique ainsi que des investigations microscopiques ont mené à une compréhension des phénomènes de transfert de masse d’une part et des effets structurels des ultrasons sur les filaments de spiruline d’autre part. Selon les résultats expérimentaux, la MTS a permis d'obtenir 229 % de protéines en plus (28,42 ± 1,15 g / 100 g MS) par rapport au procédé classique sans ultrasons (8,63 ± 1,15 g / 100 g MS). Avec 28,42 g de protéines pour 100 g de spiruline dans l'extrait, un taux de récupération des protéines de 50 % a été atteint en 6 minutes effectives avec un procédé MTS continu. Partant de ces résultats prometteurs, des pistes d’extrapolation ont été étudiées afin de proposer des outils d’aide à la décision pour l’industrialisation du procédé. Ainsi une procédure d’analyse des risques (HACCP & HAZOP), une étude de coût ainsi que l’impact environnemental du procédé ont été développés dans une seconde partie de ces travaux. Enfin, des voies de valorisation des co-produits d’extraction ont été présentées dans une approche de bioraffinerie
Microalgae are one of the most promising renewable resource for future sustainable food. Thanks to their diversity of metabolism, these microorganisms can synthesize a wide range of compounds of interest with high nutritional value. However, their consumption remains limited because of their intrinsic organoleptic characteristics unattractive. To tackle this problem and to overcome these barriers, this thesis was focused on the development of a production process of food ingredient from spirulina.A green and innovative method using ultrasonic technology for the extraction of proteins from Arthrospira platensis was proposed in a first part. This is the manothermosonication (MTS). The use of an experimental plan made it possible to optimize extraction parameters; and mathematical modeling and microscopic investigations led to an understanding of the mass transfer phenomena on the one hand, and the structural effects of ultrasound on spirulina filaments on the other hand. According to the experimental results, MTS allowed to obtain 229 % more proteins (28.42 ± 1.15 g / 100 g DW) compared to the conventional method without ultrasound (8.63 ± 1.15 g / 100 g DW). With 28.42 g of protein per 100 g of spirulina in the extract, a protein recovery rate of 50% was achieved in 6 minutes with a continuous MTS process. Based on these promising results, extrapolation tracks have been studied in order to propose decision support tools for process industrialization. Thus, a risk analysis procedure (HACCP & HAZOP), a cost study as well as the environmental impact of the process were developed in a second part of this work. Lastly, ways of exploiting by-products have been presented in a biorefinery approach

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy: Biotechnology, Durban University of Technology, Durban, South Africa, 2017.
Fossil carbon dioxide emissions can be biologically fixed which could lead to the development of technologies that are both economically and environmentally friendly. Carbon dioxide, which is the basis for the formation of complex sugars by green plants and microalgae through photosynthesis, has been shown to significantly increase the growth rates of certain microalgal species. Microalgae possess a greater capacity to fix CO2 compared to terrestrial plants. Selection of appropriate microalgal strains is based on the CO2 fixation and tolerance capability, both of which are a function of biomass productivity. Microalgal biomass could thus represent a natural sink for carbon. Furthermore, such systems could minimise capital and operating costs, complexity, and energy required to transport CO2 to other places. Prior to the development of an effective CO2 mitigation process, an essential step should be to identify the most CO2-tolerant indigenous strains. The first phase of this study therefore focused on the isolation, identification and screening of carboxyphilic microalgal strains (indigenous to the KwaZulu-Natal province in South Africa). In order to identify a high carbon-sequestering microalgal strain, the physiological effect of different concentrations of carbon sources on microalgae growth was investigated. Five indigenous strains (I-1, I-2, I-3, I-4 and I-5) and a reference strain (I-0: Coccolithus pelagicus 913/3) were subjected to CO2 concentrations of 0.03 - 15% and NaHCO3 of 0.05 - 2 g/1. The logistic model was applied for data fitting, as well as for estimation of the maximum growth rate (µmax) and the biomass carrying capacity (Bmax). Amongst the five indigenous strains, I-3 was similar to the reference strain with regards to biomass production values. The Bmax of I-3 significantly increased from 0.214 to 0.828 g/l when the CO2 concentration was increased from 0.03 to 15% (r = 0.955, p = 0.012). Additionally, the Bmax of I-3 increased with increasing NaHCO3 concentrations (r = 0.885, p = 0.046) and was recorded at 0.153 g/l (at 0.05 g/l) and 0.774 g/l (at 2 g/l). Relative electron transport rate (rETR) and maximum quantum yield (Fv/Fm) were also applied to assess the impact of elevated carbon sources on the microalgal cells at the physiological level. Isolate I-3 displayed the highest rETR confirming its tolerance to higher quantities of carbon. Additionally, the decline in Fv/Fm with increasing carbon was similar for strains I-3 and the reference strain (I-0). Based on partial 28S ribosomal DNA gene sequencing, strain I-3 was found to be homologous to the ribosomal genes of Chlorella sp. The influence of abiotic parameters (light intensity and light:dark cycles) and varying nutrient concentrations on the growth of the highly CO2 tolerant Chlorella sp. was thereafter investigated. It was found that an increase in light intensity from 40 to 175 umol m2 s-1 resulted in an enhancement of Bmax from 0.594 to 1.762 g/l, respectively (r = 0.9921, p = 0.0079). Furthermore, the highest Bmax of 2.514 g/l was detected at a light:dark cycle of 16:8. Media components were optimised using fractional factorial experiments which eventually culminated in a central composite optimisation experiment. An eight-factor resolution IV fractional factorial had a biomass production of 2.99 g/l. The largest positive responses (favourable effects on biomass production) were observed for individual factors X2 (NaNO3), X3 (NaH2PO4) and X6 (Fe-EDTA). Thereafter, a three-factor (NaNO3, NaH2PO4 and Fe-EDTA) central composite experimental design predicted a maximum biomass production of 3.051 g/l, which was 134.65% higher when compared to cultivation using the original ASW medium (1.290 g/l). A pilot scale flat panel photobioreactor was designed and constructed to demonstrate the process viability of utilising a synthetic flue gas mixture for the growth of microalgae. The novelty of this aspect of the study lies in the fact that a very high CO2 concentration (30%) formed part of the synthetic flue gas mixture. Overall, results demonstrated that the Chlorella sp. was able to grow well in a closed flat panel reactor under conditions of flue gas aeration. Biomass yield, however, was greatly dependent on culture conditions and the mode of flue gas supply. In comparison to the other batch runs, run B yielded the highest biomass value (3.415 g/l) and CO2 uptake rate (0.7971 g/day). During this run, not only was the Chlorella strain grown under optimised nutrient and environmental conditions, but the culture was also intermittently exposed to the flue gas mixture. Results from this study demonstrate that flue gas from industrial sources could be directly introduced to the indigenous Chlorella strain to potentially produce algal biomass while efficiently capturing and utilising CO2 from the flue gas.
D

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011.
Due to greenhouse gas emissions from fossil fuel usage, the impending threat of global climate change has increased. The need for an alternative energy feedstock that is not in direct competition to food production has drawn the focus to microalgae. Research suggests that future advances in microalgal mass culture will require closed systems as most microalgal species of interest thrive in highly selective environments. A high lipid producing microalga, identified as Chlorella vulgaris was isolated from a freshwater pond. To appraise the biofuel potential of the isolated strain, the growth kinetics, pyroletic characteristics and photosynthetic efficiency of the Chlorella sp was evaluated in vitro. The optimised preliminary conditions for higher biomass yield of the selected strain were at 4% CO2, 0.5 g l-1 NaNO3 and 0.04 g l-1 PO4, respectively. Pulse amplitude modulation results indicated that C. vulgaris could withstand a light intensity ranging from 150-350 μmol photons m-2s-1. The pyrolitic studies under inert atmosphere at different heating rates of 15, 30, 40 and 50 ºC min-1 from ambient temperature to 800 oC showed that the overall final weight loss recorded for the four different heating rates was in the range of 78.9 to 81%. A tubular photobioreactor was then designed and utilised for biomass and lipid optimisation. The suspension of microalgae was circulated by a pump and propelled to give a sufficiently turbulent flow periodically through the illuminated part and the dark part of the photobioreactor. Microalgal density was determined daily using a Spectrophotometer. Spectrophotometric determinations of biomass were periodically verified by dry cell weight measurements. Results suggest that the optimal NaNO3 concentration for cell growth in the reactor was around 7.5 g l-1, yielding maximum biomass of 2.09 g l-1 on day 16. This was a significant 2.2 fold increase in biomass (p < 0.005) when compared to results achieved at the lowest NaNO3 cycle (of 3.8 g l-1), which yielded a biomass value of 0.95 g l-1 at an OD of 1.178. Lipid accumulation experiments revealed that the microalga did not accumulate significant amounts of lipids when NaNO3 concentrations in the reactor were beyond 1.5 g l-1 (p > 0.005). The largest lipid fraction occurred when the NaNO3 concentration in the medium was 0.5 g l-1. Results suggest that the optimal trade-off between maximising biomass and lipid content occurs at 0.9 g l-1 NaNO3 among the tested conditions within the photobioreactor. Gas chromatograms showed that even though a greater number of known lipids were produced in Run 8, the total lipid percentage was much lower when compared to Runs 9-13. For maximal biomass and lipid from C. vulgaris, it is therefore crucial to optimise nutritional parameters such as NaNO3. However, suitable growth conditions for C. vulgaris in a tubular photobioreactor calls for innovative technological breakthroughs and therefore work is ongoing globally to address this.

Eukaryotic microalgae from the Red Sea were isolated, characterized and identified with the purpose of building a culture collection that will serve future research activities in the area of industrial microbiology. Seven sampling locations were surveyed using an in-house designed isolation protocol. Microalgae enrichment was carried out in vitro using the streak plate method and fluorescence activated cell sorting approaches. Colonial and cellular microscopy, growth media preference assays, as well as temperature, pH and salinity tolerance tests were carried out to describe the isolates. DNA extraction, PCR amplification, template sequencing and in silico analyses were carried out to identify the isolates and arrange them in a proper phylogenetic description. In total, 129 isolates were obtained. From these, only 39 were selected for characterization given their increased ability of accumulating large amounts of biomass in solid and liquid media in relatively short periods of time. All of these have a green color, are unicellular, non-motile, photosynthetic organisms and have a cell size ranging from 5 to 8 µm. More than half of them showed growth preference in Walne media, followed by F/2, MN and BG-11 SW. Maximum temperature tolerance of all organisms was around 38 ºC, while optimum growth was observed close to 25 ºC. pH preference was diverse and three groups were identified: acidic (6), intermediate (8 - 9) and alkaline (> 10) growing isolates. Salinity tests showed an overall growth preference at 25 PSU, approximately 10 units lower than that found at the sampling stations. Most isolates showed diminished growth at high salinity and high pH, except for OS3S1b which grew well in both cases, and could be an interesting strain to study further. Twenty four isolates were related to Ulvophyceae sp. MBIC10591 by BLAST approaches with a maximum identity of 96 - 97%. A maximum likelihood phylogenetic tree was created for these isolates, relative to the BLAST hits and to some model eukaryotic microalgae for positioning reference. It was shown that the 24 OS isolates are related to each other with a confidence value of 84%. Differential responses of all high biomass producing isolates towards abiotic stresses might suggest that each represents a distinct, novel, unclassified marine organism.

從微藻提煉的生物燃料，是化石燃料和其他生物燃料的優良替代品。藻類生物燃料屬碳中性，因為微藻為光自養生物，能經光合作用吸收二氧化碳，並將之轉化成碳氫化合物和脂肪。碳氫化合物和脂肪可用以提煉生物燃料。此外，微藻可以吸收廢水中的污染物作生長的營養，同時作污水處理。
本研究項目的目的為透過下述方法，降低藻類生物燃料的生產成本，並提高藻株的脂肪含量： (1) 篩選可以在污水自養培育，並有高產油量的微藻菌株，(2) 以兩階段培養方法，用處理過的污水作培養，從而提高油脂產，(3) 透過微藻毒理測試，和水質化學分析，研究處理後的污水中影響微藻生長的污染物和有毒物質。
這個研究中使用從沙田污水處理廠收集的二級處理污水，其水質亦被研究。幾種微藻菌株分別為小球藻 (Chlorella pyrenoidosa)，叢粒藻 (Botryococcus braunii) 和微綠球藻 (Nannochloropsis oculata)，從鰂魚池水分離出的小球藻 (Chlorella sp.1)，及兩種從處理污水中分離出的小球藻(Chlorella sp. 2, Chlorella sp. 3)。微藻菌株分別在培養基和處理污水中培養，並比較在兩種情況下的脂肪，脂肪酸，碳水化合物，蛋白質含量，生物質量和總有機碳。結果發現，雖然經處理的污水中營養成分非常低 (<0.11 mg / L活性磷，<9.68 mg / L硝酸根，<0.5 mg / L鉀離子)，所有研究的微藻菌株都能存活。在兩階段培養法下，首先以「氮含量充足階段」(培養基)提高生物質量，然後以「氮含量不足階段」(經處理污水) 培養，培養成本可以降低，同時提高脂肪生產率。在兩階段培養法下，叢粒藻的脂肪生產率比在人工培養基和經處理污水高2.6倍和7.13倍。
沙田污水處理廠處理的污水水質良好，並無驗出有害重金屬，雙酚A(BPA)，四溴雙酚A(TBBPA)和2,3,7,8-四氯二苯並二噁英(TCDD)。從藻類產生的生物燃料將不含有重金屬。
在這個研究中的叢粒藻 (Botryococcus braunii)，微綠球藻 (Nannochloropsis oculata)和小球藻 (Chlorella sp.1)都可以容忍雙酚A(BPA)，四溴雙酚A(TBBPA)，二氯苯氧氯酚 (TCS)和2,3,7,8-四氯二苯並二噁英(TCDD)。他們可以培育在其他來源的經處理污水。
利用經處理污水於兩階段培養法，是一種新的、更經濟的增加微藻油脂產量方法，亦可以配合任何其他方法，以減低藻類生物燃料的製造成本。
Biofuel from microalgae can be an excellent substitute of fossil fuel and other biofuels. Algal biofuel is carbon neutral as microalgae are photoautotrophic. Through photosynthesis, microalgae can capture and convert carbon dioxide to hydrocarbons or lipids which can be used for biofuel production. Besides, microalgae can use pollutants from wastewater as nutrients for growth, which can serve as a wastewater treatment process.
The aims of the project are to lower the cost of algal biofuel production and boost up lipid content of algal strains by (1) screen a microalgal strain that can be cultivated in treated sewage autotrophically and give high oil yield, (2) use two phase cultivation, with treated sewage as medium, to boost up lipid productivity, (3) investigate heavy metals and some organic pollutants that may exist in treated sewage and can affect algal growth by performing algal toxicity test and chemical analysis of treated sewage.
The secondarily treated sewage used in this project was collected from the Sha Tin Sewage Treatment Works. The quality of the secondarily treated sewage was monitored. Chlorella pyrenoidosa, Botryococcus braunii and Nannochloropsis oculata from commercial source, and Chlorella sp. 1 isolated from tilapia fish pond water, and two species of algae, Chlorella sp. 2 and Chlorella sp. 3, isolated from treated sewage were investigated. Microalgal strains are compared by investigating the content of lipid, fatty acid, carbohydrate, protein, biomass and total organic carbon when cultivated in culture medium and treated sewage. Results found that although nutrients in treated sewage were very low (<0.11 mg/L reactive phosphorus, <9.68 mg/L nitrate and <0.5 mg/L potassium ion), all the microalgae investigated could grow reasonably well. Using two phase cultivation, with an initial nitrogen sufficient phase (artificial media) for biomass production, followed by nitrogen limitation phase (treated sewage), cost of cultivation could be reduced and the overall lipid productivity could be increased. Under the two phase cultivation, the lipid productivity of Botryococcus braunii was 2.6 and 7.13 fold higher than cultivated in artificial medium and treated sewage respectively.
Treated sewage from the Sha Tin Sewage Treatment Works was in good quality without harmful concentrations of heavy metal and BPA, TBBPA and TCDD. The microalgae could not absorb or adsorb significant amount of the harmful substances and the algal biofuel produced would not contain heavy metals. All the microalgae investigated in this project could tolerate BPA, TBBPA, TCS and TCDD. They could be cultivated in treated sewage from other sources.
Two phase cultivation using treated sewage is a new way for increasing lipid productivity from microalgae economically and can be combined with any other means for producing algal biofuel with lowest cost.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Kwan, Ka Ki.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 104-113).
Abstracts also in Chinese.
Acknowledgements --- p.i
Abstract --- p.iii
摘要 --- p.Vi
Table of Contents --- p.viii
List of Figures --- p.Xii
List of Plates --- p.Xvi
List of Tables --- p.xviii
Abbreviations --- p.xx
Chapter 1. --- General introduction
Chapter 1.1 --- Fossil fuel, the major energy source nowadays --- p.1
Chapter 1.2 --- Disadvantages of using fossil fuel --- p.3
Chapter 1.3 --- Biofuel --- p.5
Chapter 1.4 --- Disadvantages of traditional biofuel production --- p.8
Chapter 1.5 --- Characteristics of microalgae --- p.9
Chapter 1.6 --- Biofuel from microalgae --- p.14
Chapter 1.7 --- Nutrients for microalgae related to lipid production --- p.18
Chapter 1.8 --- Current research on algal biofuel --- p.19
Chapter 1.9 --- Two phase cultivation as a new way for lipid production --- p.24
Chapter 1.10 --- Objectives --- p.24
Chapter 2. --- Biofuel production under two phase cultivation with artificial medium and treated sewage
Chapter 2.1 --- Introduction --- p.26
Chapter 2.2 --- Materials and Methods --- p.28
Chapter 2.2.1 --- Algal strains collection and isolation --- p.28
Chapter 2.2.2 --- Artificial culture media --- p.29
Chapter 2.2.2.1 --- Bristol’s Medium (BM) --- p.29
Chapter 2.2.2.2 --- Modified Bold 3N medium (MBM) --- p.31
Chapter 2.2.3.3 --- F/2 medium (F/2) --- p.33
Chapter 2.2.3 --- Water quality of treated sewage --- p.33
Chapter 2.2.3.1 --- Chemical and biological condition --- p.34
Chapter 2.2.3.2 --- Total organic carbon and total nitrogen (TOC/TN) --- p.35
Chapter 2.2.3.3 --- Reactive phosphate --- p.35
Chapter 2.2.3.4 --- Nitrate --- p.37
Chapter 2.2.3.5 --- Ammonia --- p.39
Chapter 2.2.3.6 --- Metal elements --- p.40
Chapter 2.2.4 --- Cultivation conditions --- p.40
Chapter 2.2.5 --- Growth monitor of microalgae in artificial medium and treated sewage --- p.41
Chapter 2.2.6 --- Comparison of microalgae cultivated in artificial media and treated sewage --- p.42
Chapter 2.2.6.1 --- Large scale cultivation --- p.42
Chapter 2.2.6.2 --- Cell morphology --- p.43
Chapter 2.2.6.3 --- Cell harvesting --- p.44
Chapter 2.2.6.4 --- Dried biomass --- p.44
Chapter 2.2.6.5 --- Lipid content --- p.45
Chapter 2.2.6.6 --- Fatty acid profile --- p.46
Chapter 2.2.6.7 --- Extraction of carbohydrates and protein --- p.48
Chapter 2.2.6.8 --- Carbohydrate content --- p.48
Chapter 2.2.6.9 --- Protein content --- p.49
Chapter 2.2.7 --- Two phase cultivation --- p.50
Chapter 2.2.8 --- Statistical analysis --- p.50
Chapter 2.3 --- Results --- p.51
Chapter 2.3.1 --- Water quality of treated sewage --- p.51
Chapter 2.3.2 --- Nutrient contents in artificial medium --- p.54
Chapter 2.3.3 --- Growth of microalgae in artificial medium and treated sewage --- p.54
Chapter 2.3.3.1 --- Cell morphology and cell size --- p.57
Chapter 2.3.3.2 --- Biomass --- p.59
Chapter 2.3.3.3 --- Lipid content --- p.61
Chapter 2.3.3.4 --- Fatty acid profile --- p.63
Chapter 2.3.3.5 --- Carbohydrates content --- p.66
Chapter 2.3.3.6 --- Protein content --- p.67
Chapter 2.3.4 --- Two phase cultivation --- p.69
Chapter 2.4 --- Discussion --- p.74
Chapter 2.4.1 --- Water quality of treated sewage and nutrients in artificial medium --- p.74
Chapter 2.4.2 --- Growth of microalgae in artificial medium and filtered treated sewage --- p.75
Chapter 2.4.3 --- Microalgae cultivated in artificial media and treated sewage --- p.76
Chapter 2.4.4 --- Two phase cultivation --- p.81
Chapter 3. --- Possible toxic effect on algal growth from chemicals in sewage
Chapter 3.1 --- Introduction --- p.84
Chapter 3.2 --- Materials and methods --- p.85
Chapter 3.2.1 --- Analysis of dissolved metals by ICP --- p.85
Chapter 3.2.2 --- Organic compounds --- p.86
Chapter 3.2.3 --- Algal bioassay --- p.87
Chapter 3.3 --- Results --- p.88
Chapter 3.3.1 --- Dissolved metals and metalloids --- p.88
Chapter 3.3.2 --- Organic compounds --- p.88
Chapter 3.3.3 --- Algal bioassay --- p.91
Chapter 3.4 --- Discussion --- p.97
Chapter 4. --- Conclusion and future prospectives --- p.99
Chapter 4.1 --- Summary --- p.99
Chapter 4.2 --- Genetic engineering --- p.100
Chapter 4.3 --- Further study --- p.102
Chapter 4.4 --- Conclusion --- p.102
Chapter 5. --- References --- p.104

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011.
The depletion of global energy supplies coupled with an ever increasing need for energy and the effects of global warming have warranted the search for alternate renewable sources of fuel such as biodiesel. First generation biofuels are not sustainable enough to meet long term global energy requirements and more recently there has been concern expressed as to the potential negative implication of crop based biofuels in the form of negative energy balances and potentially no greenhouse gas benefit due to land utilisation not being taken into account. Microalgae have shown great promise as a sustainable alternative to first generation biofuels. They have faster growth rates, have greater photosynthetic efficiencies, require minimal nutrients and are capable of growth in saline waters which are unsuitable for agriculture. Microalgae utilise a large fraction of solar energy and have the potential to produce 45 to 220 times higher amounts of triglycerides than terrestrial plants. The use of microalgae for biodiesel production requires strain selection, optimisation and viability testing to ascertain the most appropriate organism for large scale cultivation. This study focuses on bioprospecting for indigenous lipid producing microalgae, screening, selection and optimisation of growth and lipid yields with respect to nutrient limitation. Further we have ascertained the sustainability of a selected species of microalgae in open pond system. Chlorella sp. and Scenedesmus sp. were found to be dominant amongst the isolates. Strains we selected and underwent media selection and growth and lipid optimisation trials. BG11 media was selected as the most appropriate media for the growth of the selected Chlorella and Scenedesmus strains. Little variation in growth was observed for both cultures ten days into cultivation under varying nitrate concentrations. Phosphate optimum was shown to be 0.032g/l for Scenedesmus sp and 0.04g/l for Chlorella sp. Best lipid yield determined during exponential growth was achieved in cultures with 0.3g/L to 0.6g/L nitrate and phosphate as per BG11 medium. pH optimisation showed that cultures may be adapted to growth at higher pH over time. The optimum pH range for growth was determined to be narrow and was found to be between pH 10 and pH 11. Chlorella sp. was shown to be sustainable as a dominant culture in open pond system. Open pond systems however are prone to contamination by other species of microalgae within weeks of inoculation.

A biotecnologia das microalgas está a ganhar cada vez mais importância, havendo já um número significativo de estudos publicados. Contudo, a grande maioria dos trabalhos realizados continua a ser com macroalgas. A maior parte dos estudos com macro- e microalgas têm, contudo, uma base empírica, relacionada com os organismos congéneres que são as plantas vasculares. No trabalho apresentado nesta Tese revemos inicialmente algumas das aplicações das microalgas, em especial as de origem marinha. Após uma resenha sobre os polissacarídeos (capítulo 1.1), focamo-nos na aplicação (potencial) dos biocompostos na saúde humana, em geral (capítulo 1.2) e nas doenças cardiovasculares, em particular (capítulo 1.3) e ainda em doenças crónicas como as relacionadas com o stress oxidativo, a diabetes ou o Parkinson, devido às suas propriedades antioxidantes. Em seguida, aprofundamos o conhecimento sobre os polissacáridos algais (capítulo 2). Neste capítulo, revemos as características bioquímicas deste tipo de polímeros e mostramos algumas das vastas aplicações dos polissacáridos produzidos pelas microalgas (capítulo 2.1). Mostramos ainda quão abrangente pode ser a utilização destes compostos, quer em aplicações médicas (capítulo 2.2), quer em nutrição, devido à sua riqueza em fibras (capítulo 2.3). Sabendo que duas das espécies produtoras de elevadas quantidades de carotenoides são a Dunaliella salina e a Haematococcus pluvialis, desenvolvemos um primeiro trabalho experimental em que os respectivos meios de cultura foram enriquecidos com as fito-hormonas cinetina (kin) e ácido diclorofenoxiacético (2,4-D) (capítulo 3.1). A influência positiva de 2,4-D foi bastante significativa, dado que a adição de 1.0 mg /l meio de cultura de H. pluvialis, sózinha ou em conjunto com kin (1.0 mg /l), aumentou a produção de biomassa em 320% e 290% respectivamente. Uma ainda menor concentração de 2,4-D (0.5 mg /l) induziu, em D. salina (a 15% salinidade, NaCl m/v), um aumento de 410% no número de células. Num trabalho experimental posterior submeteu-se a Haematococcus a condições de stress (privação de nutrientes e luminosidade elevada) para indução da carotenogénese. A biomassa foi depois recolhida e procurou-se a melhor forma de manter a estabilidade dos carotenoides produzidos e uma taxa de degradação mais reduzida. As melhores condições de preservação verificaram-se quando se secou as algas por atomização (temperatura de entrada de 180ºC e temperatura de saída de 110ºC) e armazenamento dos pós a -21ºC em azoto. Desta forma conseguiu-se recuperar 90% do pigmento astaxantina (capítulo 4). Quando se manipulou o meio de cultura de Porphyridium cruentum, verificou-se que a adição de sulfato (21 mM em sulfato) aumentou a produção do exopolissacárido (EPS), e o conteúdo em proteína (52 mM e 104 mM em sulfato) e sulfato (104 mM) no EPS. Contudo, as características físico-químicas do polímero não sofreram alterações. Este EPS mostrou possuir capacidade antiviral, em especial contra o vírus Vesicular estomatitis (capítulo 3.2). Duas experiências diferentes permitiram mostrar que as microalgas têm também aplicação na área ambiental (capítulo 5). Verificou-se que a flora autóctone de um efluente de cervejeira pode ser utilizada para remoção de grande parte do azoto e fósforo, mas também para remover uma parte significativa da carga orgânica dessa água residual. Devido à qualidade da proteína obtida — na qual se observou um aumento de vários aminoácidos essenciais — e do perfil lipídico da biomassa — tendo-se verificado um aumento de alguns ácidos gordos como o ácido eicosapentaenóico (ou EPA) —, a biomassa final pode ser recolhida e eventualmente utilizada em rações para animais, ou ainda para produção de biodiesel (capítulo 5.1). Numa última experiência testou-se a influência da utilização das microalgas, tanto sozinhas como em consórcio com rizobactérias promotoras do crescimento, no melhoramento dos solos. Os pós das algas Chlorella vulgaris e/ou dos consórcios obtidos por atomização foram também incluídos em microcápsulas revestidas por maltodextrina (MD) e goma-arábica (GA) ou gelatina (G). Os consórcios de Chlorella:Stenotrophomonas encapsulados em MD:G e MD:GA 1:1 (m/m) foram os que induziram um melhor crescimento, em termos de biomassa seca, tanto da raiz, como da parte aérea das plantas utilizadas neste estudo (capítulo 5.2). Resumindo, pensamos que, com estes trabalhos, contribuímos para o aumento do conhecimento em biotecnologia de microalgas e teremos talvez alertado para o grande potencial das microalgas, especialmente as de origem marinha, devido à sua grande variedade e riqueza da sua biomassa em compostos bioactivos.
The biotechnology of microalgae is gaining more and more importance, and a significant number of studies is already published. However, the vast majority of the work has been performed with macroalgae. Nonetheless, most of the studies with macro- and microalgae have an empirical basis, relating to the counterpart organisms, which are vascular plants. In the beginning of the work presented in this thesis, we reviewed some of the applications of microalgae, especially the ones of marine origin. After reviewing the literature on polysaccharides (chapter 1.1), we focused on the (potential) application of the biocompounds from microalgae in human health, in general (chapter 1.2), and in the cardiovascular diseases, in particular (chapter 1.3). We also focused on the applications in chronic diseases, such as those related to the oxidative stress, like diabetes or Parkinson, due to the antioxidant properties of such compounds. Then, we went deeper into the knowledge of the polysaccharides (chapter 2). In this chapter, we reviewed the biochemical characteristics of this type of polymers, and showed some of the vast applications of the polysaccharides produced by microalgae (chapter 2.1). We showed as well how broad the uses of these compounds may be, both in medical uses (chapter 2.2) and in nutrition, due to being rich in fibre (chapter 2.3). Considering that the two species producing high amounts of carotenoids are Dunaliella salina and Haematococcus pluvialis, we developed a first experimental work on which the respective culture media were enriched with phytohormones kinetin (Kin) and dichlorophenoxyacetic acid (2,4-D) (chapter 3.1). The positive influence of 2,4-D was very significant, as the addition of 1.0 mg /l culture of H. pluvialis, alone or combined with Kin (1.0 mg /l), enhanced the production of biomass in 320% and 290%, respectively. An even lower concentration of 2,4-D (0.5 mg /l) induced an increase of 410% in the D. salina cell number (under 15% salinity, NaCl w/v). In further experimental work, we subjected Haematococcus to stress conditions (deprivation of nutrients and high brightness) to induce carotenogenesis. After collecting the carotenized biomass, we looked for the best conditions in order to maintain the stability of the produced carotenoids, with a lower degradation ratio. The best preservation conditions after spray-drying the biomass (inlet temperature of 180ºC and outlet temperature of 110ºC) showed to be the storage of the dried powders in nitrogen at -21ºC. A 90% of astaxanthin recovery was obtained at these conditions (chapter 4). During the manipulation of the Porphyridium cruentum culture medium, we verified that the addition of sulphate (21 mM in sulphate) increased the production of the exopolysaccharide (EPS), and also the protein (52 mM and 104 mM in sulphate) and sulphate content (104 mM) of the EPS. However, the physicochemical characteristics of the polymer did not suffer any modifications. This EPS showed to have antiviral properties, particularly against Vesicular stomatitis virus (chapter 3.2). Two different experiments allowed us to show that microalgae may also find application in the environmental area (chapter 5). We verified that the autochthonous flora from the effluent of a brewery might be used to remove the majority of the nitrogen and phosphorus, and also a significant part of the organic load of that wastewater. Due to the quality of the protein – where we observed an increase in several essential aminoacids — and the lipid profile of the biomass – where it was observed an increase in some fatty acids, such as eicosapentaenoic acid (EPA) —, the final biomass may be harvested and eventually used in animal feed, or to produce biodiesel (chapter 5.1). In the last experiment, we studied the influence of the use of microalgae, alone or as a consortium with growth-promoting rhizobacteria (GPRB), on soil improvement. Spray-dried powders from the alga Chlorella vulgaris and/or the consortia were also included into microcapsules coated with maltodextrin (MD) and arabic gum (GA) or gelatin (G). The consortia Chlorella:Stenotrophomonas encapsulated in MD:G and MD:GA 1:1 (w/w) were the ones that induced a higher growth, considering the dried biomass, of both the roots and the shoots of the plants used in this study (chapter 5.2). In summary, we think that, through these studies and experimental work, we have contributed to the enhancement of the knowledge in the biotechnology of microalgae and we have, at least, drawn the attention to the great potential of microalgae, particularly those from marine sources, due to their vast diversity and richness of their biomass in bioactive compounds.

Submitted in fulfillment for the requirements for the degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2015.
Main focus of this study is to investigate the enzymatic-conversion of microalgal lipids to biodiesel. However, preceding steps before conversion such as drying of microalgal biomass and extraction of lipids were also studied. Downstream processing of microalgae has several challenges and there is very little literature available in this area. S. obliquus was grown in the pilot scale open pond cultivation system for biomass production. Different techniques were studied for biomass drying and extraction of lipids from harvested microalgal biomass. Effect of these drying and extraction techniques on lipid yield and quality was assessed. Energy consumption and economic evaluation was also studied. Enzymatic conversion of microalgal lipids by extracellular and whole cell lipase application was investigated. For both applications, free and immobilized lipases from different sources were screened and selected based on biodiesel conversion. Process parameters were optimized using chosen extracellular and whole cell lipases; also step-wise methanol addition was studied to improve the biodiesel conversion. Immobilized lipase was studied for its reuse. Final biodiesel was characterized for its fuel properties and compared with the specifications given by international standards. Enzymatic conversion of microalgal lipids was compared with the conventional homogeneous acid-catalyzed conversion. Enzymatic conversion and chemical conversion were techno-economically investigated based on process cost, energy consumption and processing steps. Freeze drying was the most efficient technique, however at large scale economical sun drying could also be selected as possible drying step. Microwave assisted lipid extraction performed better compared to sonication technique. Immobilized P. fluorescens lipase in extracellular application and A. niger lipase in whole cell application showed superior biodiesel conversion. The extracellular immobilized P. fluorescens lipase showed better biodiesel conversion and yields than the immobilized A. niger whole cell lipase. Both the enzyme catalysts showed lower biodiesel conversion compared to conventional chemical catalyst and higher processing cost. However, techno-economic analysis showed that, the reuse potential of immobilized lipases can significantly improve the economics. Fewer purification steps, less wastewater generation and minimal energy input are the benefits of enzymatic route of biodiesel conversion. Microalgae as a feedstock and lipase as a catalyst for conversion makes overall biodiesel production process environmentally-friendly. Data from this study has academic as well as industrial significance. Conclusions from this study form the basis for greener and sustainable scaling-up of microalgal biodiesel production process.
D

Thesis (M.Tech.:Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xvi, 185 leaves
Spirulina is a blue-green, multicellular, filamentous cyanobacterium that can grow to sizes of 0.5 millimetres in length. It is an obligate photoautotroph and has a pH growth range from 8.3 to 11.0.The large-scale production of Spirulina biomass depends on many factors, the most important of which are nutrient availability, temperature and light. These factors can influence the growth of Spirulina and the composition of the biomass produced by changes in metabolism. Brine effluent from cooling towers of electricity generating plants may provide an ideal growth medium for Spirulina based on its growth requirements, i.e. high alkalinity and salinity. The aim of this research was to optimise brine effluent from cooling towers by supplementing it with salts, in order to use this optimised effluent in a small open laboratory raceway pond in an attempt to increase the biomass production of Spirulina.

Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Durban University of Technology, Durban, South Africa, 2015.
Fossil fuel reserves have been diminishing worldwide thus making them very scarce in the long term. These fuel sources and their by-products which are used commercially tend to produce large quantities of emissions. Some of them are believed to be toxic to flora and fauna. It is primarily for this reason that researchers worldwide have begun to seek out alternative sources of environmentally safe fuel. Biodiesel from algae is one of these sources that have been examined over the last few decades. Biodiesel has been produced from other plant-based material and waste oils in countries like America and Japan. However, the use of food based crops for biodiesel production has been challenged as it has an impact on food production on an international scale. Algae have only recently been investigated for their feasibility for biodiesel production on a large scale. The aim of this study was to investigate and develop technologies for biodiesel production from algae. The species of algae chosen were chlorella sp and scenedesmus sp., since they are indigeneous to Kwazulu Natal in South Africa. Samples were obtained from a local raceway pond and prepared for analysis. Drying protocols used freeze, oven and sun drying for initial preparation of the samples for analysis. Sun drying was the least energy intensive but most time consuming. At laboratory scale, oven drying was chosen as the best alternative. Lipid extraction methods investigated were the separating funnel method, the soxhlet method, microwave assisted extraction (MAE) and the expeller press. Thirteen solvents covering a range of polarities were used with the extraction methods to determine the efficiency of the solvent with these methods. Optimization of the MAE method was conducted using both the one factor at a time (OFAT) method and a design of experiment (DOE) statistical method. The shelf life of algal biomass was determined by ageing the samples for approximately three months. Direct and in-situ transesterification of lipid extracts to produce biodiesel was investigated using both acid and base catalysis. Qualitative and quantitative analyses were conducted using Fourier transform infra-red (FTIR) and gas chromatography (GC). Chemical and physical characterization of the biodiesel produced from the algal lipid extracts were compared to both local and international standard specifications for biodiesel. In terms of extraction efficiency, it was found that soxhlet and microwave assisted extraction methods were almost equally good. This was proved by the MAE method yielding an average of 10.0% lipids for chloroform, ethanol and hexane after 30 mL of solvent was used in an extraction time of 10 minutes, while the soxhlet method yielded 10.36% lipids using an extraction volume of 100 mL of solvent with an extraction time of 3 hours. Chloroform, ethanol and hexane were more efficient than the other ten solvents used. This was shown by these three solvents producing lipid quantities between 10% to 11% while all the other solvents produced lipid quantities between 2 and 10 %. The best extraction efficiency was achieved by the binary solvent mixture made up of chloroform and ethanol in a 1:1 ratio. Under the conditions optimized, this solvent ratio yielded a lipid content of 11.76%. The methods chosen and optimized for extraction are very efficient, but the actual cost of production of biodiesel need to be determined. Physical methods like the expeller press are not feasible for extraction of the type of biomass produced unless algae are pelletized to improve extraction. This will impact on the cost of producing biodiesel. The transesterification protocols investigated show that the base catalysis produced biodiesel with a ratio of saturates to unsaturates conducive to a good fuel product. The direct esterification method in this study proved to be better than the in-situ method for biodiesel production. The in-situ method was also more labour intensive. Chromatography was found to be a fast and efficient method for qualitative and quantitative determination of biodiesel. Characterization tests showed that the quality of biodiesel produced was satisfactory. It also showed that the methods used in this study were feasible for the satisfactory production of biodiesel which meets local and international specifications.

Nos últimos anos, verificou-se um aumento dos estudos relativos a aplicações de macroalgas e de microalgas como resultado da identificação de diversas substâncias sintetizadas por estes organismos. A imensa biodiversidade e consequente variabilidade na composição bioquímica da biomassa obtidas nas culturas de algas, aliadas ao melhoramento genético das mesmas e ao desenvolvimento de tecnologia de cultivo em grande escala, tornou-as um alvo de interesse para a indústria, nomeadamente, a indústria alimentar e farmacêutica. As algas são uma potencial fonte de obtenção de variados compostos biologicamente ativos, tais como carotenoides, ácidos gordos, vitaminas, polissacáridos, entre outros, com uma eficácia superior à verificada pelas tradicionais culturas vegetais terrestres, podendo estes ser empregues no desenvolvimento de alimentos funcionais, o que conduziu ao recente aumento do interesse comercial das algas. As propriedades naturais das algas permitem a extração de composto com atividade antibacteriana, antitumoral, antifúngica, antivírica, etc., compostos estes difíceis de sintetizar, permitindo assim maior eficiência no desenvolvimento de fármacos. Para além disso, existem atualmente no mercado diversos cosméticos, direcionados para o tratamento cutâneo e para a proteção solar, que contêm extratos de algas na sua composição.
In recent years, there have been an increasing number of studies on applications of algae and micro-algae resulted in the identification of several substances synthesized by these organisms. The immense biodiversity and the resulting variability in the biochemical composition of the biomass obtained in cultures of algae and microalgae, combined with the genetic improvement of these and the development of large-scale cultivation technology, making them a target of interest for industry, including industry food and pharmaceutical industries. The algae are a potential source for obtaining various biologically active compounds, such as carotenoids, fatty acids, vitamins, polysaccharides, among others, with greater efficacy to those seen by traditional land crops, and they may be employed in developing functional foods, which led to the recent increase in the commercial interest of algae. The natural properties of the compound of algae allow extraction antibacterial, antitumor, antifungal, antiviral, etc., these compounds difficult to synthesize, thus allowing for more efficient drug development. Furthermore, there are currently in various cosmetics market, targeted for skin treatment and sunscreen containing algae extracts in its composition.

Submitted in fulfillment of the requirements of the degree of Master of Applied Science in Biotechnology, Durban University of Technology, 2015.
In light of the world’s declining fossil fuel reserves, the use of microalgal biodiesel has come to the forefront as a potentially viable alternative liquid fuel. The depleting freshwater reserves make the feasibility of this concept questionable. The use of wastewater reduces the requirement for depleting freshwater supplies. This project aimed to determine the viability of municipal domestic wastewater effluent as a substrate for microalgal growth, in order to generate an economical and environmentally friendly source of biofuel. Wastewater effluents from three domestic wastewater treatment plants were characterized in terms of known microalgal nutrients viz., ammonia, phosphate and nitrates. Phosphate concentrations varied throughout the year and were found to be low (< 3 mgL-1) whilst ammonia and nitrate concentrations ranged from 0 to 10 mgL-1 throughout the experimental period. These wastewaters were found to be suitable for cultivating microalgae. The study explored the cultivation of Chlorella sorokiniana on pre- and post-chlorinated domestic wastewater effluent to assess their potential as a medium for high microalgal culture density and lipid production. Post-chlorinated wastewater effluent was found to be superior to pre-chlorinated wastewater effluent, as evident by the higher biomass concentration. This wastewater stream did not contain high concentrations of bacteria when compared to pre-chlorinated wastewater effluent. Nitrogen is an essential nutrient required for regulating the growth and lipid accumulation in microalgae. Cultures growing in post-chlorinated effluent had a lifespan of 18 d. Residual nitrogen in wastewater effluent supported microalgal growth for limited periods. Supplementation using cheap, readily available nitrogen sources was required for optimal biomass and lipid production. Urea, potassium nitrate, sodium nitrate and ammonium nitrate were evaluated in terms of biomass and lipid production of C. sorokiniana. Urea showed the highest biomass yield of 0.216 gL-1 and was selected for further experimentation. Urea concentrations (0–10 gL-1) were assessed for their effect on growth and microalgal physiology using pulse amplitude modulated fluorometry. A concentration of 1.5 gL-1 urea produced 0.218 gL-1 biomass and 61.52 % lipid by relative fluorescence. Physiological stress was evident by the decrease in relative Electron Transport Rate from 10.45 to 6.77 and quantum efficiency of photosystem II charge separation from 0.665 to 0.131. Gas chromatography analysis revealed that C16:0, C18:0, C18:1, C18:2 and C18:3 were the major fatty acids produced by C. sorokiniana. Wastewater effluent has been considered an important resource for economical and sustainable microalgal biomass/lipid production. The study showed that C. sorokiniana was sufficiently robust to be cultivated on wastewater effluent supplemented with urea. The results indicate that supplemented wastewater effluent was an acceptable alternative to conventional media. Using a relatively cheap nitrogen source like urea can certainly improve the techno-economics of large scale biodiesel production.