Microalgae are gaining more attention for several reasons such as being potential producers of sustainable fuel, for use as health supplements and in skincare. Simris Alg is a Swedish company that produces Omega 3 supplements from a primary producer of these fatty acids - the algal diatom Phaeodactylum tricornutum, which is a sustainable alternative to Omega 3 derived from fish. Omega 3 fatty acids constitute a small fraction of the total algal biomass, and to increase profitability and utilize all of the biomass, the purpose of this thesis project is to present potential applications for the residual material that is left after oil extraction. A general composition study was made of Simris Alg algal residue material, and results are compared to those found in previous studies of P. tricornutum biomass. An optimization of the fractionation is needed to separate the storage carbohydrate chrysolaminarin and cell wall component glucuronomannan, followed by analysis for confirmation. Also, it would be interesting to separate chitin, if there is any, since despite the presence of chitin synthases, it is unclear whether the diatom actually produces chitin. When gathering information, no actual experimental characterization of carbohydrate active enzymes involved in synthesis of the main carbohydrates investigated were found. Such information would be useful to increase production of the carbohydrate of interest, if valuable applications are found. Potential applications of various cell components, such as carbohydrates, in skincare would be interesting to investigate, as well as optimizing fucoxanthin extraction for use as an additional high value product next to Omega 3.

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I detta arbete undersöks möjligheterna att använda mikroalger som råvara för produktion avbiogas, både genom en litteraturstudie och genom en förstudie för hur en pilotanläggning förhållbar odling av mikroalger för biogasproduktion skulle kunna initieras. Utgångspunkten ärhållbarhet, vilket innebär att odlingen av mikroalgerna baseras på befintliga, outnyttjadesamhällsflöden. Avloppsvatten och koldioxidutsläpp är exempel på flöden som kan orsakanegativ miljöpåverkan i form av klimatförändringar, övergödning och försurning. Om dessaresurser istället får utgöra närings- och kolkällor vid odling av mikroalger för produktion avbiogas utnyttjas dessa resurser istället till att generera hållbart producerad energi. Närbiogasen ersätter fossila bränslen ökar miljövinsten ytterligare. Det är dock grundläggande attproduktionen är ekonomiskt hållbar, och litteraturstudien visar att detta inte är fallet vidmikroalgbaserad produktion av endast biogas. Därför krävs tillämpning av ettbioraffineringskoncept, där inte bara biogas produceras av mikroalgerna utan även andraprodukter som till exempel vätgas, enzymer, värme och elektricitet. I förstudien beskrivs hurett projekt kan planeras för att utreda om ett sådant system kan bli hållbart. Det delas upp ifyra delprojekt: 1) systemdesign och hållbarhetsanalys; 2) en projektplan för delprojekt 3 och4; 3) laboratorietester; och 4) tester i pilotskala. Endast det första delprojektet, där systemetsom beskrivs i litteraturstudien definieras och hållbarhetsanalyseras, diskuteras i detalj iförstudien. Genomförandet av delprojekten ingår inte i detta arbete. / This paper examines the possibility of using microalgae as raw material for the production ofbiogas. This was achieved through studies of relevant literature as well as through a basicproject plan regarding the initiation of a pilot plant. The theory of sustainable microalgaecultivation is based on the utilization of existing resource flows that are currently unutilized insociety, such as waste water and flue gas emissions. These resources can cause environmentalissues such as climate change, eutrophication and acidification. However, they can alsoprovide the nutrients necessary for effective microalgal growth, and the microalgae can inturn be utilized as a sustainable energy source for production of biogas. Replacing fossil fuelswith microalgal biogas may lead to environmental benefits. A review of literature, however,shows that when biogas alone is produced from the microalgae the process is noteconomically sustainable. Hence, a biorefinery concept is suggested where products such ashydrogen, enzymes, heat and power make the system sustainable. A basic project plandiscribes one possible scenario for the initiation of sustainable cultivation of microalgae andthe subsequent biorefining process for the production of biogas. The project is divided intofour phases: 1) system design and sustainability analysis; 2) a feasibility study for phase 3 and4; 3) laboratory tests; and 4) pilot scale tests. Only the initial phase of the project, whichdefines the system design described in the literature study and provides a sustainabilityanalysis over the defined system, is discussed in detail. None of the project phases will beimplemented as part of this study.

Microalga-based technologies are introduced to provide a new generation of biofuels. The cultivation of microalgae in wastewater as the growth medium may offer the dual benefit of wastewater treatment and sustainable biofuel production. The potential of microalgal biomass to be converted into biodiesel depends on the accumulation of lipids, mainly triacylglycerols. However, the presence of toxic contaminants in municipal or industrial wastewater could negatively affect the biomass growth and modify the pattern of lipid accumulation in algae cells. This research aims to evaluate the potential of cultivating microalgae in wastewater for the removal of selected contaminants and the production of biodiesel. The hypothesis underlying this work was that growing microalgae in the presence of contaminants, typically found in wastewater, may induce a stress response that potentially increases the lipid productivity and enhances the biofuel yield. This research explored the potential of microalgae to be used for the removal of wastewater pollutants while accumulating high concentrations of neutral lipid that can be converted into biodiesel. Physiological and transcriptional responses of two genera of green algae were observed in the presence of stresses induced by toxic metals in raw hydraulic fracturing flowback water and pharmaceuticals found in municipal wastewater. Two green algae, Dunaliella salina and Chlorella vulgaris, were selected based on the availability of genetic information, current trends in the alga industry, and specific growth requirement in hypersaline flowback water and municipal wastewater. The first part of this research focuses on the possibility of using the salt-tolerant green microalga, D. salina, for the removal of toxic metals from hydraulic fracturing flowback water. In the second part of this research, the freshwater green microalga, C. vulgaris, was exposed to a suite of pharmaceuticals, commonly found in municipal wastewater, and the effects on the biomass growth and lipid accumulation were evaluated. In Chapter 3, aqueous geochemical characteristics of hydraulic fracturing flowback water were determined. Metagenomic analysis was also used to characterize the microbial community structure of the water samples. This technique included DNA extraction, PCR amplification of genes for 16S and 18S ribosomal RNA, and pyrosequencing. Although the two flowback water samples were shown to have water property measures in the range of reported values in literature, they were significantly different from each other regarding water properties such as total dissolved solids and total organic carbon. They also had distinct microbial community structures. It was shown the flowback water samples contained mainly halophilic, anaerobic, and thermophilic species with a pattern that was expected to be seen in natural gas shale reservoirs. Chapter 4 covers the characterization of the effects of contaminants (toxic metals) in hydraulic fracturing flowback water on the biomass growth and cellular lipid accumulation in Dunaliella salina. Metals were analyzed using ICP-MS. Biomass accumulation was determined by spectrophotometry and gravimetric methods. Lipid accumulation and composition were determined by GC-MS after conversion into fatty acid methyl esters. D. salina was successfully cultivated in saline hydraulic fracturing flowback water, and algal biomass growth was associated with a significant reduction of several toxic metals in the samples. D. salina, grown in flowback water, accumulated high levels of cellular lipids. Also, the estimated biodiesel properties were comparable with other algal biodiesel and complied with the US and EU biodiesel standards. The last chapter of part one presents the effects of a collection of metals commonly found in hydraulic fracturing flowback water and other industrial wastewaters on growth and lipid accumulation of D. salina. The impacts of the nitrogen concentration in the growth medium were also observed. The Nile red assay (fluorescence measurement after Nile red staining) was used to assess lipid accumulation. A range of toxic effects on the algal growth was observed. However, the effects were lower at higher nitrogen level that may indicate to an improved toxicity resistance when nutrient stress is minimal. Even with a high dose-dependent toxicity on growth, some toxic metals increased lipid accumulation. Chapter 6 describes the microplate-based toxicity test conducted to characterize the effect of a range of emerging contaminants (pharmaceuticals) commonly found in municipal wastewater on the biomass growth and cellular lipid accumulation measured by Nile red staining assay. Similar to what was observed for metals in Chapter 5, some pharmaceuticals increased lipid accumulation, even with a high dose-dependent toxicity on algal growth. Most tested pharmaceuticals at municipal wastewater levels (low) did not have a significant effect on the algal growth and lipid accumulation. Toxicity data (EC50 of growth) were well in agreement with what was reported in literature and antibiotics were the most toxic compounds among the different pharmaceutical classes. Chapter 7 presents a further characterization of the effects of a few selected compound from the previous chapter. Changes in the algal lipid composition upon exposure to the selected compounds were determined by qualitative and quantitative analysis of the extracted lipids using GC-MS. FT-IR spectroscopy was used to detect changes in the cellular constituents. The transcriptional responses of the key genes involved in the lipid metabolism of C. vulgaris were also investigated upon exposure to tetracycline using reverse-transcription real-time PCR and the results were interpreted based on the measured levels of cellular lipids and other metabolites. Lipid accumulation was changed upon exposure of algae to pharmaceuticals; however, the FAMEs profile did not show a significant variation. FT-IR spectroscopy highlighted the subtle changes in the cellular basis, and FT-IR peak associated with lipids was strongly correlated with total FAMEs quantified by GC-MS (Pearson correlation coefficient = 0.89). Gene expression analysis showed a general down-regulation of genes involved in the lipid biosynthesis. Results from this research are expecting to help develop and optimize innovative algal-based technologies combining the benefits of wastewater treatment, sustainable biofuel production, and mitigation of carbon dioxide emissions. / Civil Engineering

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.

This thesis project is a contribution to addressing the pressing need for sustainable alternatives to fossil fuels and traditional plastics. The work focuses on enzymatic saccharification of microalgae biomass to generate a sugar-rich stream as raw material for biofuels and bioplastics production. Microalgae and cyanobacteria are highlighted for their ability to convert CO2 into valuable biomass components in the presence of light.  To enhance biomass conversion efficiency, the enzymatic hydrolysis of microalgae and cyanobacteria is explored by utilizing various enzymes to break down complex polymers into valuable sugars. Additionally, the cultivation of cyanobacteria is studied to optimize the overall process. Results indicate challenges, such as measurement uncertainty and the need for biomass pretreatment, suggesting areas for further research.  The primary objective of this thesis work is to optimize enzymatic hydrolysis processes by determining the optimal combinations of enzymes, to enhance biomass conversion efficiency. It also underscores the importance of microorganisms in transitioning to a more environmentally friendly future by offering sustainable alternatives to conventional products.

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.

Microbes, living on the boundary between the sediment and the water-column in lakes, can play a pivotal role in governing the magnitude and frequency of nutrient cycling. The purpose of this research was to focus on the role of benthic microalgae in regulating such processes and to identify spatial and temporal characteristics in their function. Approaches included the quantification of sediment nutrient concentrations (particularly P fractionation), estimates of equilibrium phosphate concentrations (EPC0) (resuspended and undisturbed sediment estimates), and assessment of the benthic microalgal community composition, biostabilisation capacity, and its ability to regulate diffusive-nutrient flux. This thesis highlighted the importance of biological regulation of benthic/pelagic nutrient cycling, especially the role of benthic microautotrophs. Release sensitive sediment-P fractions were observed to be highly variable (both with depth and season) and correlated well with indicators of benthic photosynthesis (e.g. DO, chlorophyll, pH). Understanding the seasonality of whole-system P partitioning can enhance future lake management programmes. EPC0 estimates were significantly higher during undisturbed as opposed to disturbed sediment conditions. Epipelon constituted < 17 % of the total sediment chlorophyll signal and was highest in the clearer winter months and at intermediate depths at which a trade off between wind-induced habitat disturbance and light limitation existed. In intact core experiments, the benthic microalgal community significantly reduced the diffusive nutrient (especially PO₄-P and SiO₂) flux. NH₄ -N release was highest under light conditions at high temperatures. The mechanisms for regulation included direct uptake, photosynthetic oxygenation of the sediment surface, and regulation of nitrification/denitrification processes. Sediment stability increased with colloidal carbohydrate concentration (extruded by benthic microbes) at 4.1 m water-depth but not at 2.1 m overlying water depth, probably indicating the role of habitat disturbance in shallow areas acting to reduce epipelic production. Additionally, in an ecosystem comparison, the nature and extent of the biotic mediation of sediment stability varied between freshwater and estuarine ecosystems.

Sécuriser un approvisionnement fiable de micro-algues est récemment devenu un enjeu industriel. Pour assurer la croissance de micro-algues dans des photobioréacteurs clos, un transfert de masse optimum de l'oxygène et du dioxyde de carbone doit être assuré. Dans cette thèse, une étude du transfert de masse gaz-liquide dans les conduites horizontales a été menée. Dans les trois premiers chapitres, un modèle unidimensionnel de transfert de masse dans le photobioréacteur a été développé. Tout d'abord, le transfert de masse entre une bulle de gaz allongée et un écoulement liquide turbulent a été 'étudié expérimentalement. En considérant l'interface comme étant plane, les coefficients de transfert de masse mesurés sont proches d'une corrélation de Lamourelle (1972). Le modèle de Taitel pour les écoulements stratifiés a été comparé à des modèles plus complets pour la prédiction de l'interface des bulles allongées. Une approche analytique basée sur un bilan de masse et utilisant les modèles testés a ensuite été développée et adaptée à un photobioréacteur afin de prédire l'efficacité de la conversion du CO2 en biomasse en fonction des paramètres d'exploitation. Les deux derniers chapitres visent à développer une simulation numérique du transfert de masse gaz-liquide. Une mesure de la concentration en CO2 dans le sillage d'une bulle de gaz ascendante a été effectuée à l'aide d'une méthode améliorée de traitement des données de Fluorescence Induite par Plan Laser (FIPL). Enfin, une simulation numérique a été réalisée sous COMSOL / Securing a reliable supply of microalgae has recently become an industrial stake. To ensure successful growing of microalgae in enclosed, tubular photobioreactor systems as in Microphyt, an optimum mass transfer of oxygen and carbon dioxide should be secured. In this thesis an investigation of the gas-liquid mass transfer in horizontal pipes has been conducted. In the first three chapters, a one dimensional mass transfer model in horizontal gas-liquid flows will be developed and applied to horizontal photobioreactors. Firstly, a study of mass transfer between an elongated gas bubble under a turbulent liquid flow immobilized in a duct has shown that under the hypothesis considering the interface as a flat plane estimated, the measured mass transfer coefficients appear to be well fitted by a correlation from Lamourelle (1972). The interface prediction for stratified flows have been compared to more complete unit-cell models for intermittent flow interface and found to be a good first estimate. The photobioreactor’s conversion efficiency of CO2 into biomass as a function of operating parameters is investigated using an analytical approach to complete the mass balance and classical two-phase flow approach from Taitel (1976). The last two chapters aim at developing a numerical simulation of gas-liquid mass transfer. A measurement of CO2 wake structures behind free rising bubbles have realized using an improved data treatment method for Planar laser-induced fluorescence (PLIF) and pH sensitivity of fluorescein sodium. Finally, an implementation of the experimental measurements under COMSOL has been realised