Growing interest in biofuel production from non-fossil fuel sources has resulted in several studies exploring different raw material sources as feedstock, including many algae species, for large-scale production of biofuel. Algae are promising feedstock due to advantages such as its short growth cycle, high biomass production, and lipid content. However, there are still challenges to overcome in order to use algae for commercial biofuel production. One of these challenges is the requirement for a large quantity of water and nutrients needed for growing large quantities of the algae. This work explores a potential solution to this challenge by studying the possibility of using industrial wastewater to grow algae for biofuel production. However, many industrial wastewaters, including effluents from semiconductor processing plants, are known to contain heavy metals that are toxic to humans and the environment. In this work, the effects of four of such metals ions, As(V), As(III), Ga(III), and In(III) on Chlorella sorokiniana and Scenedesmus obliquus strains were studied. In particular, the heavy metal toxicity on the strains, effects on its growth rate, biomass yield, lipid content and fatty acid methyl esters (FAME) were studied. Also, the effect of controlling pH on growth rate, biomass yield, lipid content, and FAME was studied for Chlorella sorokiniana in the presence of Ga(III). The results of the study confirmed the toxicity of these metals on both strains. However, Ga(III) and In(III) had the highest effect, while As(V) showed the least toxicity to the strains, with Chlorella sorokiniana withstanding concentrations of As(V) as high as 140mg/L. The heavy metals were slightly more toxic to Scenedesmus obliquus compared to Chlorella sorokiniana. In addition, the heavy metals reduced the growth rate of both strains. High percent changes in growth rate (more than 50%) were seen in cultures containing Ga(III) and In(III). Furthermore, concentration measurements with Inductively Coupled Plasma Optical Emission Spectrometer (ICP) before, during, and at the end of the growth period, showed that Scenedesmus obliquus adsorbed higher amounts of the heavy metals compared to Chlorella sorokiniana. Microalgae biosorption of heavy metals limits its end use, hence making Scenedesmus obliquus a less favorable option for this study, but may be a better choice for wastewater treatment applications. The effects of the four metals on the lipid content and FAME profile of Chlorella sorokiniana were studied. The result showed an increase in Chlorella sorokiniana lipid content in the presence of In(III), but a decrease in the presence of As(V) and As(III). The heavy metals had effects on the strain’s FAME compositions. The fatty acid composition included C16:0, C16:1, C16:2, C16:3, C18:0, C18:1, ω-6, C18:2, ω-6, and C18:3, ω-3 accounting for more than 97% of the total FAME composition. Furthermore, controlling the pH of the culture in the presence of Ga(III) at 6.5 led to higher adsorption of the heavy metal, increase in lipid content, but no significant change in FAME composition.

Silver nanoparticles (AgNPs) are a promising nanomaterial with numerous applications and high level of commercialization. Biomass-mediated AgNP synthesis has emerged as a novel approach for producing AgNPs and microalgal biomasses have been found particularly advantageous. However, few studies have so far focused on microalgae-mediated biosynthesis and the mechanism of AgNP biosynthesis is still elusive. The purpose of this study was twofold: 1) to investigate effects of different parameters on the biosynthesis of AgNPs; 2) to investigate the mechanisms involved in such a bioprocess. It was found that the cell extract of Neochloris oleoabundans prepared by whole cell aqueous extraction (WCAE) in boiling water bath was able to reduce Ag+ to AgNPs. It was further discovered that sonication of algal cells before extraction could enhance the efficiency of cell extraction and enable AgNP biosynthesis using cell extract obtained by disrupted cell aqueous extraction (DCAE) at room temperature. Light was required for AgNP biosynthesis and rainbow tests showed that purple and blue lights were particularly necessary. Based on experimental results, we hypothesize the mechanism of microalgae-mediated AgNP synthesis to be a chlorophyll-mediated reaction, in which chlorophylls are excited upon absorbing photons in the purple and blue spectra and donate electrons to reduce Ag+, the lost electrons are replenished by water-splitting reaction.

Philosophiae Doctor - PhD / Biofuel production from microalgae is currently not economically competitive with fossil fuels due to high operational costs. A sustainable system needs to be developed which considers cultivation, harvesting and conversion to fuels as a single loop. The harvesting step has been identified as a major bottleneck within the biofuel production process, contributing to a significant proportion of the operational cost (20-30%). Chemical flocculation is a more affordable alternative to centrifugation and filtration. Chemical flocculants however negatively impact the quality of biomass and conversion efficiency to biofuel by increasing biomass ash content. Bioflocculation with biopolymers or microbes have a minimal impact on the quality of biomass. In this study, the interaction between the filamentous fungus Isaria fumosorosea and the microalgae C. sorokiniana is investigated. Under strict autotrophic conditions at pH 7-8, co-culture of microalgae (2-20 μm) with fungal blastospores resulted in theidevelopment of large pellets (1-2 mm) which may be easily harvested by sedimentation or filtration at 95% harvesting efficiency. Fungal assisted bioflocculation was compared to other harvesting methods with respect to cost and impact on the hydrothermal conversion process. Low cost carbon sources, including waste hydrothermal nutrients, minimal sugar concentrations and algal exudate may reduce fungal cultivation costs. Waste products, such as organic carbon, N, P, CO₂ and trace metals can be recycled and used for algae and fungal cultivation, closing the loop to make the system sustainable. / National Research Foundation; Swiss Government

Biodiesel from microalgae is a viable alternative for replacing the global demand for petro-diesel. High biomass and lipid production are key desirable characteristics needed in a species to be used for biodiesel production. It has been demonstrated in literature that the increase in salinity can increase the lipid content of microalgae, but lower the growth rate of a species. Therefore the effect that salinity has on the growth and lipid content of ten microalgal species, isolated from a warm temperate solar saltworks, was investigated. The microalgae were cultivated at a temperature of 22°C and at salinities ranging from 17 to 70 psu. It was found that growth and lipid production for all species were influenced to some degree by the salinity. Growth rates greater than 0.6 d-1 showed a decrease with higher salinity. Most (71 percent) of the growth rates that exceeded 0.6 per day were exhibited by cultures exposed to normal salinity (35 psu). This shift is a good indication that salinity inhibits/slows down growth and that the species in general prefer lower salinity conditions. Growth rates ranged from 0.17 ± 0.05 to 1.19 ± 0.17 d-1. Lipid content for the diatoms (2.78 ± 0.36 to 10.86 ± 4.59 percent DW) were lower than expected, whereas the lipid content for the green flagellates (3.10 ± 1.56 to 22.64 ± 1.19 percent DW) was on par with that reported in literature. To bring results into perspective a production model was developed to simulate a production scenario at the Swartkops Saltworks. Lipid and productivity results obtained in this study were used to estimate how much oil and biomass can be produced within the ponds of the Swartkops Saltworks. The model showed that although microalgae cultivation for biodiesel is technically feasible, at present it is not economically viable to do so.

Orientadores: Maria Regina Wolf Maciel, Luiz Fernando de Lima Luz Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-24T13:41:08Z (GMT). No. of bitstreams: 1 RiosPinto_LuisaFernanda_D.pdf: 4599056 bytes, checksum: 80a3191a923471837068d7b5f547c19e (MD5) Previous issue date: 2014 / Resumo: Atualmente, a destruição ambiental devido à exploração inapropriada dos recursos naturais não renováveis, o aquecimento global, e a contaminação da biosfera causada, principalmente, pela globalização e um crescimento tecnológico acelerado, tem levado ao desenvolvimento de diversos combustíveis de fontes de energia renováveis, que sejam capazes de manter ou superar a eficiência dos hidrocarbonetos fósseis. Neste cenário, se deu o desenvolvimento do biodiesel, um combustível biodegradável derivado de fontes renováveis, o qual, na sua combustão, libera menos gases tóxicos, como são os óxidos de enxofre e nitrogênio. O biodiesel pode ser produzido a partir de gorduras animais, óleos vegetais ou biomassa. A fim de reduzir os custos da produção do biodiesel, matérias primas como os lipídios vêm sendo estudadas. Neste contexto, as microalgas oferecem uma série de vantagens como fonte de lipídios, como por exemplo, altas taxas fotossintetizantes, alta produção de biomassa e um rápido crescimento, se comparado, principalmente, com outras fontes de óleo vegetal. Neste trabalho, foi realizado o estudo do crescimento da microalga Desmodesmus sp. sob condição autotrófica, mixotrófica e heterotrófica com o objetivo de obter maior teor de lipídios na célula da microalga, visando a produção de biodiesel. Nas condições autotróficas, se obteve um crescimento de 53,1 Mcélulas mL-1. No cultivo mixotrófico, conseguiram-se 181 Mcélulas mL-1 e no heteretrófico atingiu-se 298 Mcélulas mL-1, todos no sexto dia de cultivo. As porcentagens de lipídios foram de 23%, 25% e 17% (m/m) da biomassa para o cultivo autotrófico, mixotrófico e heterotrófico, respectivamente. O óleo extraído do cultivo autotrófico, foi caracterizado via cromatografia gasosa e, a partir desta análise, produziu-se um óleo sintético (mistura de óleos vegetais e animais) com as características semelhantes da microalga para realizar a cinética da reação de transesterificação, num reator em batelada. A produção de biodiesel foi feita por transesterificação ácida, com excesso de etanol. O tempo de reação foi de 24 horas e obteve-se 90% de ésteres produzidos / Abstract: Currently, environmental destruction caused by inappropriate exploitation of non-renewable natural resources, global warming, and pollution of the biosphere caused mainly by globalization and accelerated technological growth has led to the development of various fuels from renewable source, which are able to maintain or exceed the effectiveness of fossil hydrocarbons. Biodiesel, a biodegradable fuel derived from renewable source, which releases less toxic gases such as sulfur and nitrogen oxides in its combustion. Biodiesel can be obtained from animal fats, vegetable oils or biomass. In order to reduce the cost of biodiesel production, raw materials such as lipids have been studied. In this context, microalgae offer a number of advantages as a source of fat, such as high photosynthetic rates, high biomass, and fast growth rate, compared to other sources of vegetable oil. The objective of this work was to study the autotrophic, mixotrophic and heterotrophic growths of microalgae Desmodesmus sp in order to optimize the levels of lipids in the microalgae cell to produce biodiesel. In autotrophic conditions, it was observed 5.31x107 cells ml-1; in mixotrophic were achieved 1.81x108 cells ml-1; and in heterotrophic reached 2.98x108 cells ml-1 were reached after sixth day of cultivation. The percentages of lipids of biomass for autotrophic, heterotrophic mixotrophic cultivation were 23%, 25% and 17% (m/m) respectively. Oil extracted from autotrophic cultivation was characterized via gas chromatography. A synthetic oil (mixture of vegetable and animal oils) with similar characteristics to the microalgae was used to perform the transesterification kinetics in a batch reactor. The production of such biodiesel was performed under acid conditions with excess of ethanol for 24 hours yielding 90% product / Doutorado / Desenvolvimento de Processos Químicos / Doutora em Engenharia Quimica

L’océan domine la surface de notre planète et joue un rôle majeur dans la régulation de notre biosphère. Par exemple, les microorganismes photosynthétiques vivant dans l’océan produisent 50% de l’oxygène que nous respirons tous les ans, et une grande partie de notre alimentation et des ressources minérales en proviennent. En cette époque de crise écologique liée à l’accumulation anthropogénique de gaz à effet de serre dans l’atmosphère, il est impératif de développer des énergies plus durables que les carburants fossiles. Le biodiesel pourrait être une source de carburant viable et durable pour remplacer le pétrodiesel mais jusque-là, nos efforts visant à produire des lipides à base de microalgues se sont essentiellement concentrés sur des algues vertes. Dans cette thèse je propose des méthodes pour mieux caractériser une autre catégorie de microalgue : les diatomées. Les diatomées sont une composante importante du phytoplancton et contribuent 40% de la production marine de biomasse primaire. Les diatomées accumulent des lipides en réponse à la carence en nutriments, et même si elles semblent accumuler tout autant de lipides que les microalgues vertes, les voies métaboliques menant à l’accumulation de lipides sont encore peu connues.Dans cette thèse je décris notre caractérisation du glycerolipidome de la diatomée modèle Phaeodactylum tricornutum ainsi que notre étude du remodelage de lipides suite à la carence d’azote ou de phosphate. Des accessions de P. tricornutum isolées dans différentes régions de l’océan ont aussi été étudiées afin de comparer leurs réponses au stress nutritif. Nous avons trouvé que la réponse métabolique menant à l’accumulation de lipides en carence d’azote ou de phosphate est différente. En effet, la carence en azote semble déclencher le recyclage des galactoglycerolipides chloroplastiques ainsi qu’une augmentation de la biosynthèse de novo d’acides gras, alors que la carence en phosphate est plus sévère car nous avons observé une accumulation plus significative de triacylglycerols ainsi que la déplétion totale des phospholipides. De plus, nous avons observé des réponses au stress différentes entre les accessions de P. tricornutum, et en particulier concernant leur capacité à accumuler des lipides. Nous proposons l’hypothèse que ces différences sont liées à leur aptitude à recycler du carbone issu de molécules de stockage non lipidiques.Des approches génomiques ont permis de nombreuses avancées pour mieux comprendre le métabolisme des lipides de microalgues mais notre compréhension des voies de biosynthèse de lipides chez les diatomées est encore limitée. Il y a eu diverses tentatives de caractérisation de la réponse au stress de carence par approche transcriptomique mais l’étude de ces données est incomplète du fait de l’annotation insuffisante des gènes encodant les voies métaboliques pertinentes. Ainsi, dans cette thèse je décris nos tentatives d’annotation de gènes impliqués dans le métabolisme des lipides de P. tricornutum ainsi que les approches d’ingénierie génétique visant à mieux caractériser certains de ces gènes. J’ai également utilisé notre nouvelle annotation de gènes impliqués dans le métabolisme des lipides pour effectuer une étude comparative de plusieurs transcriptomes de P. tricornutum en conditions de carence trouvés dans la littérature. J’ai ainsi pu produire une liste de gènes potentiellement impliqués dans l’accumulation de lipides. Enfin, nous avons pu utiliser ces données pour aider l’interprétation de données génomiques et transcriptomiques issues de la diatomée oléagineuse Fistulifera solaris afin de mieux comprendre comment elle accumule des quantités importantes de lipides pour des applications dans le secteur des biotechnologies et des bioénergies. / The ocean dominates the surface of our planet and plays a major role in regulating the biosphere. For example, the microscopic photosynthetic organisms living in the ocean provide 50% of the oxygen we breathe every year, and much of our food and mineral resources are extracted from the ocean. In a time of ecological crisis linked to the accumulation of anthropogenic greenhouse gases in the atmosphere, we must investigate more sustainable energies than fossil fuels. Much attention has been given to biodiesel but so far most efforts to efficiently produce triacylglycerols in microalgae have focused on green algae. In this thesis I propose approaches to better understand another type of microalgae that is significantly divergent from green lineages: diatoms. Diatoms are a major phylum of phytoplankton in the ocean and account for 40% of marine primary productivity. While diatoms appear to be at least as effective as green algae for producing lipids, the fatty acid and glycerolipid biosynthetic pathways leading to their production have not yet been well characterized. Therefore, I propose to better characterize these pathways in the model diatom Phaeodactylum tricornutum in order to help unlock the potential of diatoms for lipid-based biotechnological applications.In this thesis, I discuss our attempts to establish a reference for the glycerolipidome of P. tricornutum and of our assessment of the lipid remodeling and accumulation that occurs in response to nitrogen- and phosphorus-starvation. A range of accessions of P. tricornutum isolated from different parts of the ocean were also examined to compare their responses to nutrient deprivation. We found that the metabolic response leading to lipid accumulation in different nutrient-deprived conditions are distinct. Nitrogen-deprivation appears to trigger the recycling of chloroplastic galactoglycerolipids as well as a strong increase in de novo fatty acid synthesis while the response to phosphorus-deprivation was more severe as we observed a higher triacylglycerol pool and the complete depletion of phospholipids. Furthermore, we observed several differences among accessions of P. tricornutum regarding their ability to accumulate triacylglycerol in response to nutrient starvation and propose the hypothesis that these differences are linked to their ability to recycle intracellular carbon from non-lipid storage molecules.Genome-enabled approaches have also allowed significant steps towards elucidating the lipid metabolism of microalgae in the past decade, but our understanding of diatom metabolic pathways is still limited compared to that of other microalgae and higher plants. There have been several attempts to characterize the stress response in P. tricornutum by using transcriptomic approaches but this data is difficult to exploit to its full potential without a better annotation of genes encoding the relevant pathways. Therefore, in this thesis I discuss our attempts to annotate P. tricornutum lipid metabolism genes. Based on this annotation I have attempted to better characterize a selection of genes by genetic engineering and have pursued a comparative study of several published transcriptomes of P. tricornutum in nutrient deprived conditions to produce a list of candidate genes likely to be involved in triacylglycerol accumulation. Finally, we used this data to help interpret genome and transcriptome data of the newly sequenced oleaginous diatom Fistulifera solaris to help understand how it accumulates unusually high amounts of triacylglycerol for applications in the biotechnology and bioenergy industry.

The presented diploma thesis deals with the analysis and production of selected microbial metabolites of the yeast Rhodotorula kratochvilovae in comparison to various species of microalgae. The theoretical part summarizes the knowledge about the conditions for optimal growth and production of secondary metabolites. Analytical methods, by which metabolites were quantified and evaluated, were also described. In the experimental part of this thesis, the amount of accumulated biomass, exoglycolipids, intracellular lipids, phycobiliproteins and carotenoids according to various nutrient sources in the culture medium was studied. Specifically, the yields of these metabolites were investigated in the utilization of various sources of nitrogen (yeast extract, urea, ammonium sulfate, ammonium chloride and potassium nitrate), carbon (glucose, mannose, xylose, glycerol and lactose) and at variable C / N ratios (20; 40; 70; 100 and 120: 1) ideally selected sources of observed nutrients in partial experiments. The last part of the experiment was focused on comparison of the analysis of metabolites of autotrophic algae (chlorophylls, phycobiliproteins and lipids).

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22763号 / 工博第4762号 / 新制||工||1745(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 高岡 昌輝, 教授 清水 芳久, 准教授 大下 和徹 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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.

Les secteurs de la parfumerie et de la cosmétique occupent une place proéminente dans la société moderne. De nombreuses entreprises se positionnent depuis plusieurs années sur les produits cosmétiques à ingrédients naturels. Les plantes, longtemps considérées comme matière première principale pour le domaine de la cosmétique, sont aujourd’hui concurrencées par les microalgues dont la biomasse devient plus facile à obtenir grâce aux avancées en biotechnologie bleue. Ainsi, Cosmo International Ingredients se positionne à travers cette thèse pour élargir son panel de matières premières valorisables dans le domaine de la cosmétique. Dans un premier temps, l’étude phytochimique de microalgues péruviennes a permis d’isoler et identifier une famille majeure de métabolites chez les microalgues : les glycolipides. Une recherche de conditions d’extraction optimale pour cette famille a été effectuée et a permis de proposer une méthodologie verte, spécifique et peu coûteuse. Les cyanobactéries connues pour leur production de métabolites structurellement diversifiés ont été sélectionnées pour la culture suivant des critères spécifiques. Cette approche a permis d’isoler et de caractériser 5 composés à forte valeur ajoutée dont 4 peptides et un alcaloïde. Enfin, un forçage métabolique a été effectué sur Microcystis aeruginosa afin d’optimiser la production des 4 peptides cibles. Il en est ressorti que les paramètres température et intensité lumineuse jouent un rôle important dans la production peptidique / The sectors of fragrances and cosmetics play a prominent role in the modern society. During the last decades, several companies have been focusing on nature to provide innovative products. Plants have historically been considered the main raw material in the cosmetic field but, recently, microalgae have been identified as a worthy competitor due to the facility to obtain biomass. Thus, the company Cosmo International Ingredients supported this PhD. thesis to broaden their range of raw materials that can be used for the cosmetic industry. First, the phytochemical study of Peruvian microalgae allowed the isolation of a major family of metabolites: glycolipids. An environmentally-friendly, selective and low-cost method for their extraction from the biomass has been developed. Cyanobacteria known for their production of structurally diverse metabolites have been selected for culture following specific criteria; as a result 5 compounds have been isolated and fully characterized, 4 of which were peptides and one was an indole alkaloid. Finally, to optimize the production of the targeted bioactive peptides, a kinetic study was performed for 3 different temperatures and 3 different light intensities. These parameters were found to play a critical role for the peptide production