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.

Extraction of oil and gas in Utah’s Uintah Basin results in large quantities of wastewater, or produced water, with nutrients and residual organic chemical that represent a significant resource for producing energy-related and value-added products. Produced water was obtained as a biomass producing nutrient source from industries operating in Utah’s Uintah Basin. Within the Uintah Basin (defined as Uintah and Duchesne Counties within Utah) approximately 93 million barrels of water were produced in 2013 while only 11% of the water was disposed of through evaporation, with the national average at 2%. The rest is reinjected into the subsurface. The goal of this project was to design a system that utilizes produced water as a nutrient source for growing microalgae biomass in a biofilm form using a Rotating Algal Biofilm Reactor (RABR). The biomass would then be harvested and converted into biocrude oil using hydrothermal liquefaction (HTL). The objectives were to (1) cultivate biomass on produced water, (2) optimize the reactor to reduce energy costs to operate while increasing biomass productivity, and (3) increase feedstock quality for HTL. The RABR was constructed out of polystyrene disks, and experimentation was carried out to optimize rotational speed of the reactor. Two strains of algal biomass were identified as biofilm formers and grown using produced water as the nutrient source. The biomass was then utilized as a HTL feedstock that gave an average yield of 34.5% ash free dry weight.

Growing demand for energy worldwide has increased interest in the production of renewable fuels, with microalgae representing a promising feedstock. The large-scale feasibility of microalgae based biofuels has previously been evaluated through technoeconomic and environmental impact assessments, with limited work performed on resource requirements. This study presents the use of a modular engineering system process model, founded on literature, to evaluate the nutrient (nitrogen and phosphorus) and carbon dioxide resource demand of five large-scale microalgae to biofuels production systems. The baseline scenario, representative of a near-term large-scale production system includes process models for growth, dewater, lipid extraction, anaerobic digestion, and biofuel conversion. Optimistic and conservative process scenarios are simulated to represent practical best and worst case system performance to bound the total resource demand of large-scale production. Baseline modeling results combined with current US nutrient availability from fertilizer and wastewater are used to perform a scalability assessment. Results show nutrient requirements represent a major barrier to the development of microalgae based biofuels to meet the US Department of Energy 2030 renewable fuel goal of 30% of transportation fuel, or 60 billion gallons per year. Specifically, results from the baseline and optimistic fuel production systems show wastewater sources can provide sufficient nutrients to produce 3.8 billion gallons and 13 billion gallons of fuel per year, corresponding to 6% and 22% of the DOE goal, respectively. High resource demand necessitates nutrient recovery from the lipid-extracted algae, thus limiting its use as a value-added co-product. Discussion focuses on system scalability, comparison of results to previous resource assessments, and model sensitivity of nutrient and carbon dioxide resource requirements to system parameter inputs.

Microalgae have been so far identified as the major producers of organic matter through their photosynthetic activities. In the present work, Nannochloris sp. and Amphora sp., two marine microalgae, have been investigated for proteins and lipids production. Protein fraction was quantified using Bicinchoninic acid (BCA) assay. Protein content in Nannochloris sp. was 16.69 ±4.07 % of dry mass and in Amphora sp. it was 39.89 ±2.09 % of dry mass. Enzyme assays were conducted spectrophotometrically. Nannochloris sp. had malate dehydrogenase, peroxidase and catalase activities. Amphora sp. exhibited malate dehydrogenase, catalase and cytochrome C oxidase activities. These enzymes have several valuable applications in some metabolic pathways and as antioxidant nutrition additives. Besides, lipid extraction was conducted using methanol/ chloroform solvent extraction. Crude lipid extract was analyzed using gas chromatography-mass spectrometry. Lipid contents were 8.14 ±3.67 % in Nannochloris sp. and 10.48 ±1.26% on dry basis in Amphora sp., respectively. Nannochloris sp. fatty acids were composed of C16:0 and C18:0 that are valuable for biodiesel production, and É-3 C18:3, É-6 C18:2, É-6 C16:2 having great nutritional values. In Amphora sp., the fatty acids consisted of C14:0, C16:0 and C16:1 shown to be valuable for biodiesel production and É-3 C22:6 having high nutritional values. Furthermore, a single step conversion of microalgal oil to fatty acid methyl esters was carried out starting directly from lyophilized microalgae. This promising process, in situ transesterification, led to better yields of methyl esters as compared to conventional lipid extraction followed by separate transesterification. / Master of Science

Les alcanes et les alcènes sont des hydrocarbures non cycliques important dans l’industrie. Ils sont synthétisés à partir d'acides gras par une grande variété d’organismes mais les connaissances à ce sujet sont très limitées chez les microalgues. Le but de ces travaux était donc de rechercher la présence d’alcanes ou d’alcènes dans diverses microalgues modèles, et d’essayer d’identifier la ou les enzymes responsables de la synthèse de ces composés. Nous avons mis en évidence la présence d’hydrocarbures linéaires en C15-C17 chez les microalgues Chlorella et Chlamydomonas. Ces composés étaient synthétisés uniquement en présence de lumière. L’absence dans le génome de ces microalgues d’homologues de gènes codant pour des enzymes connues de synthèse d’alcanes/alcènes a permis de conclure à la présence d’un nouveau système de synthèse d’hydrocarbures. Des purifications enzymatique et des analyses protéomique ont permis d’identifier une enzyme candidate qui exprimée chezE. coli est suffisante à la synthèse d’hydrocarbures. L'étude de cette enzyme révella qu'il s'agissait d'une photoenzyme utilisant l'énergie des photons bleue pour décarboxyler les acides grass en alca(e)ne. La structure de cette photoenzyme montre la présence un tunnel hydrophobe contenant l’acide gras et le cofacteur FAD. Cette nouvelle enzyme nommée « alcane photosynthase » amène de nombreuses question: qu'elle est la fonction des hydrocarbures chez ces microorganismes? Quel est le mécanisme catalytique de l’alcane photosynthase? Enfin, elle offre de nouvelles possibilités pour la production de biocarburants utilisant directement l’énergie solaire. / Alkanes and alkenes are important in industry. Alkanes and alkenes are synthesized from fatty acids by a variety of organisms, such as plants and insects. However, the presence in microalgae of enzymes converting fatty acids into hydrocarbons has been poorly studied. The aim of this work was to investigate the presence of alkanes and alkenes in various microalgae models, and try to identify the enzymes responsible for the synthesis of these compounds.We have first demonstrated the presence of linear hydrocarbons C15-C17 in microalgae Chlorella and Chlamydomonas. Then we have shown that the main hydrocarbon formed in Chlorella and Chlamydomonas was derived from cis-vaccenic acid and was synthesized only in the presence of light. Absence of homologues of genes coding for known alkane/alkene biosynthetic enzymes in the genome of Chlorella and Chlamydomonas indicate the presence of an unknown pathway. Enzymatic purification and proteomic analysis allowed to identify a candidate enzyme which, expressed in E. coli lead to the formation of hydrocarbons with variable chain lengths, thus demonstrating that it was really an synthase alkane. Characterization showed that the enzyme was a photoenzyme, which used blue light to catalyse the decarboxylation of fatty acid to an alka(e)ne. The three-dimensional structure of this enzyme revealed a hydrophobic tunnel containing the fatty acid and the FAD cofactor.

There is an increasing need for renewable energy sources to replace fossils fuels which accumulate harmful byproducts in the environment. Biodiesel emits less gaseous pollutants than diesel. There are various sources for biodiesel but they are unable to meet the existing demands for fuel. Microalgae are a promising source for biodiesel because of its relatively faster growth rate, availability, and lipid content. Microalgae (JC and BT) growing in local water bodies were collected, selected on section media containing antibiotics, and used for characterizations. Experiments were conducted to study and evaluate the optimum growing conditions. Results show that both JC and BT attain maximum growth with shaking and additional aeration compared to control microalgae Dunaliella salina, Nannochlorposis oculata which do not require additional aeration for optimal growth. Lipid extraction results suggest that JC (9.7%) and BT (4.1%) have slightly higher lipid content compared to control algae e.g. Chlamydomonas reinhardtii (3.1%).

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. / National Research Foundation.

Microalgae synthesize and store large volumes of lipids (potentially over 25% of dry weight) which could provide a renewable source of biodiesel. Traditional extraction techniques often produce poor lipid yields particularly from microalgae with robust cell walls. This project investigated the role of power ultrasound as a cell disruption step in lipid extraction from four microalgal species. Nile Red staining was used to assess the time when ultrasound induced increased membrane permeability in each species and lipids were extracted using an ultrasound assisted Bligh and Dyer extraction method. A 20 kHz probe system (40% amplitude, 0.086 W/cm3) caused increased lipid recovery from dry biomass in all cases; D. salina (no cell wall) from 15 to 22.5% of dry biomass after 1 minute (26% when stressed with 35 g/L NaCl). C. concordia (thin cell wall) from 7.5 to 10.5% of dry biomass after 2 minutes (27% with 25% nitrogen reduction in growth media). N. oculata (thick cell wall) from 6.5 to 10% of dry biomass after 16 minutes (31.5% when stressed with 30 g/L NaCl). The stressed cultures yield could be improved to 35% when ultrasound was combined with S070 beating beads. Chlorella sp. (thick cell wall) from 6.3 to 8.7% of dry biomass, after 16 minutes (44% was achieved when harvested at day 9 instead of 15). A Dual Frequency Reactor (16 and 20 kHz, 0.01 W/cm3) flow system with S070 beads demonstrated that high lipid extraction yields could be achieved on a larger level with N. oculata. After 4:48 minutes sonication 24% lipid recovery was achieved. This system could theoretically increase daily microalgal oil production from 3.96 to 5.76 L per day when compared to conventional techniques, at an extra production cost of only 2.9 p/litre (1.5% increase). D. salina, N. oculata and C. concordia resumed normal growth following sonication at 20 kHz after 1-20 days (8 minutes treatment for D. salina, 60 minutes treatment for N. oculata and 16 minutes treatment for C. concordia). It was found that the supernatant of sonicated D. salina and C. concordia when added to established cultures were able to boost their growth.

Vampirovibrio chlorellavorus (Gromov et Mamkaeva, 1980) is a member of the phylum cyanobacteria that has been described as an obligate pathogen of several of the green microalga, Chlorella. It utilizes as yet unknown functions to access the contents of individual Chlorella sp. host cells, which results in cell death. Its presence in a cultivated Chlorella sorokiniana culture was first discovered using polymerase chain reaction (PCR) to amplify the 16S ribosomal DNA gene, followed by DNA sequencing. Its continued routine detection throughout much of the cultivation season suggested it was an endemic member of the phycosphere community in this open cultivation system, located in Tucson, Arizona. Ultimately, its presence resulted in rapid death of C. sorokiniana in open pond systems and reduced biomass harvest. PCR analysis of total DNA isolated from sand and soil layers removed from a nearby riverbed indicated that V. chlorellavorus resides naturally in the riverbed. The ability to manage this bacterial pathogen in cultivated Chlorella host species is hindered by the limited information available in the literature regarding the biological and genomic characteristics of V. chlorellavorus. The objective of this study was to identify environmental factors that trigger the apparent increased growth rate of V. chlorellavorus and rapid algal death during the cultivation cycle. In laboratory experiments, V. chlorellavorus was shown to cause death of C. sorokiniana when the temperature exceeded 28°C, whereas, algal death was not observed when the temperature was 24°C or lower, among the temperatures tested. Also, the bacterium was more pathogenic to C. sorokiniana, grown in open cultivation systems during the summer months, compared to the cooler season months. Futhermore, when C. sorokiniana and V. chlorellavorus were co-cultivated in the presence of sodium chloride ranging from 0-10 g per liter, the growth of the bacterium was not impeded to any extent that might suggest C. sorokiniana was rendered less susceptibility to pathogen attack. Future work involves examining more triggers and ways to inhibit V. chlorellavorus growth.

Historically, the presence of microalgae (algae hereafter) in natural waters has been viewed as a nuisance due to its adverse impact on water quality. More recently, however, algae are being investigated as potential sources of biofuel and a range of natural products. These applications require the development of large-scale cultivation systems for mass production that include growth, harvesting, concentration, and product recovery components. While challenges still remain with respect to many of the processes involved in mass production, one of the most technically and economically challenging steps is harvesting the algae from dilute growth cultures, especially in systems where chemical additives are of concern either within the algae concentrate or the effluent water. For this reason, a pH-induced flocculation/deflocculation method using the hydroxides of alkali or alkaline earth metals (e.g., lime, caustic soda) is of particular interest for algae harvesting as Na, Ca and Mg are typically present in natural waters. The goal of this research was to determine the underlying mechanisms responsible for algae coagulation by magnesium and calcium and to evaluate the potential of these mechanisms for harvesting algae for a range of synthetic and field source water chemistries. In the first two phases of this research, the mechanisms for coagulation with magnesium and calcium were studied independently. A series of bench-scale experiments were designed to isolate the potential mechanisms of algae destabilization associated with each of these cations as a function of water chemistry, and microscopic analyses were performed to characterize the flocculated algae/precipitate mixtures. In the third phase of this research, removal of algae in field source waters was evaluated with respect to the underlying science elucidated in the previous phases. The results indicate that the dominant algae destabilization mechanism associated with magnesium shifts from Mg adsorption/charge neutralization to Mg(OH)2(S) precipitation-enhanced coagulation with increasing pH. Moreover, dissolved Mg2+ adsorption to the algae surface led to effective algae coagulation, while minimizing the mass of precipitated Mg(OH)2(S). For Ca, this research identified the importance of the nucleation process (heterogeneous vs. homogeneous nucleation) on algae removal efficiency. Heterogeneous nucleation is a key factor for optimizing algae removal; thus, the degree of oversaturation with respect to CaCO3(S) is a crucial operating parameter. This research demonstrated that the algae harvesting process using pH-induced flocculation/deflocculation method can be optimized for a wide range of source waters if the water chemistry (e.g. pH, ion concentration, alkalinity, ionic strength) is properly incorporated into the system design. / text