Theses and Dissertations at Montana State University (MSU)
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Item Algal biofilms and lipids: bicarbonate amendment and nitrate stress to stimulate lipid accumulation in algal biofilms(Montana State University - Bozeman, College of Engineering, 2022) Rathore, Muneeb Soban; Chairperson, Graduate Committee: Brent M. Peyton; This is a manuscript style paper that includes co-authored chapters.Algal biofuels are compounds obtained by transesterification of algal lipids to fatty acid methyl esters (FAMEs) which can be used as biodiesel. Algal biofilms have a potential for commercial applications of algal biomass for biofuel production and provide concentrated biomass requiring less water removal to reduce biofuel production costs. Lipid production in algal biofilms is low as compared to planktonic algal growth systems and strategies for enhancing lipid content in algal biofilms need to be developed. The overarching goal of the studies presented herein was to develop lipid accumulation strategies in algal biofilms using nutrient stresses to increase triacylglycerides (TAGs) and FAMEs. First, a reactor was designed for photoautotrophic biofilm growth incorporating a novel algal biomass harvesting mechanism. Chlorella vulgaris biofilm growth was demonstrated to establish the reactor characteristics under three different inorganic carbon regimes and the presence of excess calcium to facilitate biofilm attachment and accumulation. Excess calcium resulted in precipitate formation and increasing ash content in biomass and caused difficulty in biofilm detachment. However, the highest biomass accumulation was observed in the bicarbonate and the bicarbonate with calcium treatments. Second, two different algal strains were tested for lipid accumulation under two nutrient conditions: nitrate limitation and bicarbonate addition. Algal strains included, an extremophilic freshwater diatom RGd-1, a Yellowstone National Park (YNP) isolate, and oleaginous chlorophyte C. vulgaris. High bicarbonate content at low nitrate concentration in the bulk medium provided the highest lipid accumulation as determined by Nile Red fluorescence and Gas Chromatography Mass Spectrometry (GCMS) analysis of extracted FAMEs (7-22 % wt/wt). For prevention of biomass loss and quick response to nutrient stresses to stimulate lipid accumulation, the growth medium was exchanged after initial biofilm accumulation and operated in batch mode. This was implemented to quickly introduce nutrient stresses using fresh medium to vary bicarbonate and nitrate concentrations as needed. Thus, the work presented here demonstrated enhanced lipid production in algal biofilms with nitrate stress and bicarbonate amendment is a viable strategy to increase lipid accumulation. Increased lipid content may help offset the cost for biodiesel production with more lipid product and lower processing requirements for water removal.Item Evaluation of nitorgen and carbon supplementation strategies for optimizing biomass generation during cultivation of Chlorella sorokiniana, strain SLA-04(Montana State University - Bozeman, College of Engineering, 2021) Jackson, Matthew Clifford; Chairperson, Graduate Committee: Catherine KirklandAlgal cultivation requires significant nitrogen and carbon inputs, which are expensive and can offset benefits associated with biofuel production. This research investigates growth of an alkali-tolerant Chlorella sorokiniana, strain SLA-04, using different nitrogen and carbon regimes to improve physiological knowledge of this novel organism, and improve biomass production and resource demand. Nitrate, ammonium, and urea were used efficiently by SLA-04, however pH changes during utilization of nitrate and ammonium impacted inorganic carbon availability (species and concentration). Generation of OH- during use of nitrate increased pH, increasing mass transfer of CO 2 into solution and increasing the ratio of HCO 3-/CO 2. Ammonium utilization resulted in proton generation, lowering pH and inhibiting growth. When bicarbonate, rather than CO 2, was provided, productivity improved for the urea and mixed nitrogen conditions. This likely resulted from upregulation of genes related to nitrogen and carbon assimilation in the presence of bicarbonate, however Na + cotransport with urea and nitrate is required in some organisms. It is possible that Na + was insufficient when CO 2 was provided, but not in conditions with bicarbonate since it was added as NaHCO 3-. The impact of Na +, as well as other ions, on nitrogen and carbon utilization is not well understood, but it may alter gene regulation. Bicarbonate and CO 2 both promoted increased growth relative to cultures without inorganic carbon supplementation. The highest productivities were observed when carbon supplementation, either as continued CO 2 augmentation to the air sparge or as a 50mM bicarbonate amendment, was provided during nitrogen deplete growth. Glucose availability improved productivity for conditions without CO 2 supplementation. The use of urea or a combination of nitrogen sources with bicarbonate, instead of CO 2, was promising due to (a) the low cost of urea, relative to the other nitrogen sources; (b) the potential for using wastewater containing a mix of nitrogen sources; and (c) the low cost and easy transport of bicarbonate. Future research should evaluate changes in SLA-04 gene expression resulting from the supply of different nutrients, including nitrogen and carbon sources, as well as other ions essential for growth.Item Solid acid catalysts for biomass and sugar upgrading to furans(Montana State University - Bozeman, College of Engineering, 2019) Romo, Joelle Elise; Chairperson, Graduate Committee: Stephanie Wettstein; Nathan V. Bollar, Coy J. Zimmermann and Stephanie G. Wettstein were co-authors of the article, 'Conversion of sugars and biomass to furans using heterogeneous catalysts in biphasic solvent systems' in the journal 'ChemCatChem' which is contained within this thesis.; Ting Wu, Xinlei Huang, Jolie Lucero, Jennifer L. Irwin, Jesse Q. Bond, Moises A. Carreon and Stephanie G. Wettstein were co-authors of the article, 'SAPO-34/5A zeolite bead catalysts for furan production from xylose and glucose' in the journal 'ACS omega' which is contained within this thesis.Platform chemicals derived from biomass provide a viable alternative to petroleum-based fuels, chemicals, and materials. The efficient production of chemical building blocks, such as 5-hydroxymethylfurufral (HMF) and furfural, requires an optimized catalyst and reaction system, as well as an efficient system in which catalysts and products can be easily recovered. While homogeneous acid catalysts have historically been a popular choice for furan production, additional safety, material, and corrosion considerations motivates the exploration of heterogeneous solid acid catalysts. Furthermore, biphasic reaction systems, which use an organic solvent to continuously extract products, have shown increased furan yields over aqueous and monophasic systems and can allow for easy product recovery if the boiling point is selected carefully. One class of heterogeneous catalysts known as zeolites, has unique potential for furfural and HMF production with its controlled acidic and structural properties. A novel SAPO- 34/5A zeolite bead is presented in this thesis, showing promise in catalyst design for activity, product selectivity, and stability. The combination of optimized solvent systems with carefully designed solid acid catalysts lays a framework for the progression of platform chemical production from biomass. Additionally, a comprehensive review of heterogeneous catalysts for furan production in biphasic systems is presented here, which informs decisions on optimized solvent selection.Item Fungal production of biofuel and flavor compounds in liquid and solid state(Montana State University - Bozeman, College of Engineering, 2017) Schoen, Heidi Renee; Chairperson, Graduate Committee: Brent M. Peyton; Kristopher A. Hunt, Gary A. Strobel, Brent M. Peyton and Ross A. Carlson were co-authors of the article, 'Carbon chain length of biofuel- and flavor-relevant volatile organic compounds produced by lignocellulolytic fungal endophytes changes with culture temperature' submitted to the journal 'Mycoscience' which is contained within this thesis.; Brent M. Peyton and W. Berk Knighton were co-authors of the article, 'Rapid total volatile organic carbon quantification from microbial fermentation using a platinum catalyst and proton transfer reaction-mass spectrometry' in the journal 'AMB express' which is contained within this thesis.; W. Berk Knighton and Brent M. Peyton were co-authors of the article, 'Volatile organic compound production at varying oxygen conditions in a solid state fungal reactor' submitted to the journal 'Bioresource Technology' which is contained within this thesis.; W. Berk Knighton and Brent M. Peyton were co-authors of the article, 'Production of volatile organic compounds under varying nitrogen conditions by ascocoryne sarcoides' submitted to the journal 'Biotechnology and Bioengineering' which is contained within this thesis.Oil reserves are limited, so new sources of fuels and petroleum byproducts must be found. Some endophytic, filamentous fungi produce fuel and flavor relevant compounds from minimally pretreated cellulosic materials. Additionally, fungal volatile organic compounds can act synergistically as mycofumigants to inhibit bacteria, insects, and fungi. This dissertation identifies and quantifies fungal volatile organic compounds. A new method was created to quantify the total volatile organic carbon in the gas phase. A platinum catalyst was used to completely oxidize organic compounds to carbon dioxide, which was then measured with a carbon dioxide detector. This method agreed to within 94% of volatile organic carbon measurements taken with proton transfer reaction-mass spectrometry. Additionally, fungal production of fuel and flavor relevant volatile organic compounds was measured with varying pH and temperature in liquid cultures from Nodulisporium isolates EC, CO and TI. Production was also measured for TI when grown in solid state on the agricultural byproduct beet pulp at varying oxygen conditions. Finally, the model volatile organic compound producing organism Ascocoryne sarcoides was grown in liquid state with varying nitrogen sources, including amino acids. The three Nodulisporium isolates produced longer carbon number compounds at lower temperatures, which are better biofuels and are more likely to be bioactive. This trend was especially strong among volatile organic compounds associated with fatty acid metabolism. The fungi produced fewer compounds at lower pH. In solid state, TI had the highest total production of ethanol and carbon number four and higher compounds under anoxic conditions, but the highest production rates under microaerophilic conditions. Additionally, ethanol appeared to be the only major anoxic fermentation byproduct. Finally, A. sarcoides produced the most ethanol and carbon number four and higher compounds in the gas phase with ammonium chloride as the nitrogen source. Nitrogen sources that are reactants for volatile organic compounds, like leucine and phenylalanine, had lower gas phase concentrations of volatile organic compounds.Item The stoichiometry of nutrient and energy transfer: from organelles to organisms(Montana State University - Bozeman, College of Engineering, 2016) Hunt, Kristopher Allen; Chairperson, Graduate Committee: Ross Carlson; James P. Folsom, Reed L. Taffs and Ross P. Carlson were co-authors of the article, 'Complete enumeration of elementary flux modes through scalable, demand-based subnetwork definition' in the journal 'Bioinformatics' which is contained within this thesis.; Ashley E. Beck was an author and Hans C. Bernstein and Ross P. Carlson were co-authors of the article, 'Interpreting and designing microbial communities for bioprocess applications, from components to interactions to emergent properties' in the journal 'Biotechnology for biofuel production and optimization' which is contained within this thesis.; Ryan deM. Jennings, William P. Inskeep and Ross P. Carlson were co-authors of the article, 'Stoichiometric modeling of assimilatory and dissimilatory biomass utilization in a microbial community' in the journal 'Environmental microbiology' which is contained within this thesis.; Ryan deM. Jennings, William P. Inskeep and Ross P. Carlson were co-authors of the article, 'Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community' submitted to the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.; Natasha D. Mallette, Brent M. Peyton and Ross P. Carlson were co-authors of the article, 'Theoretical and practical limitations of hydrocarbon production for a cellulolytic, endophytic filamentous fungus' submitted to the journal 'Metabolic engineering' which is contained within this thesis.All life requires the acquisition and transformation of nutrients and energy, driving processes from cellular nutrient flow to planetary biogeochemical cycling. However, the organisms and communities responsible for these processes are often uncultivable and too complex to observe directly and understand. Stoichiometric modeling, a systems biology approach, analyzes the reactions in an organism and incorporates data from multiple sources to extract biologically meaningful parameters, such as theoretical limits of conversion and yields of a metabolism. These limits and yields quantify relationships between organisms to establish governing principles, from resource requirements to community productivity as a function of population composition. The presented work expanded the stoichiometric modeling algorithm and identified fundamental principles that govern nutrient and energy transfer associated with heterotrophy, community composition, and intracellular compartmentalization. A scalable routine capable of analyzing complex metabolic networks by dividing them into tractable subnetworks was demonstrated for a eukaryotic diatom. The metabolic model contained approximately two billion routes through the network and established an international benchmark for elementary flux mode analysis. Additionally, a heterotrophic archaeon was examined for the resource requirements while consuming 29 different forms of biomass derived dissolved organic carbon. These resource requirements and limitations establish a basis to analyze heterotrophy with regard to the limiting nutrient in a variety of systems. The resulting resource requirements of heterotrophy were incorporated into a community where an iron oxidizing autotroph was hypothesized to be the primary source of carbon and energy. Analysis of the community model and in situ measurements of iron and oxygen utilization indicated additional electron donors were required to account for the observed acquisition of nutrients in some communities. Finally, limits and resource requirements for fungal production of hydrocarbons were identified as a function of carbon and energy partitioning using simulated genetic modifications, providing context regarding endophytic production of bioactive molecules for host resistance as well as endophyte capacity as a petroleum producing alternative.Item Use of bicarbonate salts in algal growth for enhancement of lipid content(Montana State University - Bozeman, College of Engineering, 2016) Pedersen, Todd Christian; Chairperson, Graduate Committee: Brent M. Peyton; Robert D. Gardner, Robin Gerlach and Brent M. Peyton were co-authors of the article, 'Assesment of Nannochloropsis gaditana growth and lipid accumulation with enhanced inorganic carbon delivery' submitted to the journal 'Journal of applied phycology' which is contained within this thesis.; Robin Gerlach, Brent M. Peyton, Gregory L. Helms and Robert D. Gardner were co-authors of the article, 'Monitoring chlorella vulgaris metabolism during bicarbonate induced lipid accumulation using 1 H high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy' submitted to the journal 'Algal research' which is contained within this thesis.Production of biofuel from microalgae has thus far been cost prohibitive due in part to expenses associated with providing the necessary nutritional requirements for growth of the algal culture. In particular, inorganic carbon must be supplied in higher concentrations than available atmospherically to achieve high density cultures necessary for biofuel production strategies. Cost of algal biomass, as a feedstock, will be the limiting factor to the realization of algal biofuels moving forward. Prior research has demonstrated bicarbonate to enhance lipid content in select algal cultures grown under stress conditions, such as nitrogen depletion. This phenomenon has come to be known as bicarbonate-induced lipid accumulation, colloquially known as 'bicarbonate triggering', and has unrealized potential in executing economical and productive algal biofuel. Still, this method has only been demonstrated in select microalgal species and relatively little metabolic information is available regarding its use. Here, two species were investigated with the use of bicarbonate salts for algal growth and lipid accumulation. Nannochloropsis gaditana is a marine microalga which produces relatively high lipid content under nutrient stressed conditions, and has not been thoroughly studied under use of bicarbonate. This organism was studied under bicarbonate supplementation in batch photobioreactor systems. Chlorella vulgaris is a fresh water green alga which has received attention as a biofuel candidate, due to high growth rates and lipid content. This organism was investigated under bicarbonate supplementation during nitrogen depletion with the use of high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy over 38 hours with a 14:10 diel cycle. N. gaditana showed best growth rates under pH controlled growth strategies during nitrogen replete conditions, and bicarbonate was seen to increase lipid content following nitrogen depletion when paired with this growth strategy. However, N. gaditana may not be an ideal candidate for biofuel production as it has relatively low growth rates compared to other industrially relevant organisms, and demonstrated low productivity in batch systems. Metabolite investigation in C. vulgaris revealed large incorporation of inorganic carbon from bicarbonate amendments into biomass, specifically monitored as increases to the biological sucrose pool and subsequent synthesis of fatty acids as carbon storage compounds.Item Biofuel production by two green microalgae utilizing wastewater and recycled nutrients for growth(Montana State University - Bozeman, College of Engineering, 2014) Halverson, Luke Daniel; Chairperson, Graduate Committee: Robin GerlachMicroalgae have received considerable attention in recent years as a viable feedstock for biofuel production. In order for algal biofuel to displace a significant amount of traditional fossil-based fuel, non-potable water must be used to avoid additional stress on dwindling freshwater supplies. Additionally, adequate nutrient sources must be available, and productive, robust strains need to be utilized. The work performed towards this thesis aims to determine the ability of two green microalgae and one diatom to grow and produce biofuel and biofuel precursors in untreated wastewater. Scenedesmus sp. strain WC-1 (WC-1), Chlorella sp. strain SLA-04 (SLA-04) and Navicula sp. strain RGd-1 (RGd-1) were initially screened for growth in untreated primary clarifier effluent and diluted anaerobic digestate. WC-1 and SLA-04 were able to grow in each condition, but RGd-1 was unable to sustain growth. After WC-1 and SLA-04 displayed successful growth, experiments were performed attempting to increase the lipid content in WC-1 and SLA-04 by varying the form and concentration of dissolved inorganic carbon present in the cultures. The addition of supplementary inorganic carbon did not increase cellular triacylglyceride (TAG) content as expected, but WC-1 and SLA-04 were able to achieve considerable fatty acid methyl ester (FAME) content. The final experiments conducted toward this thesis involved the use of recycled harvest water amended with anaerobic digestate for nutrients. WC-1 grew without inhibition during the first two generations of growth in recycled harvest water, but high ammonium concentrations due to an error during the addition of anaerobic digestate in the third generation caused reduced growth rates. SLA-04 was able to grow without inhibition during each of the three generations of growth in recycled harvest water. The results of this work may strengthen the outlook of microalgae's potential as a biofuel feedstock. Identifying robust algae that can utilize low-cost nutrients while requiring minimal supplies of freshwater is a large step towards the commercialization of algal biofuels.Item Volatile fuel & organic compound production by Ascocoryne sarcoides : exploration of environmental variables and analytical methods(Montana State University - Bozeman, College of Engineering, 2013) Mallette, Natasha Dawn; Chairperson, Graduate Committee: Brent M. Peyton; W. Berk Knighton, Gary A. Strobel, Ross P. Carlson and Brent M. Peyton were co-authors of the article, 'Resolution of volatile fuel compound profiles from Ascocoryne sarcoides: a comparison by proton transfer reaction-mass spectrometry and solid phase microextraction gas chromatography-mass spectrometry' in the journal 'AMB express' which is contained within this thesis.; Elle M. Pankratz, Albert E. Parker, Gary A. Strobel, Scott C. Busse, Ross P. Carlson and Brent M. Peyton were co-authors of the article, 'Evaluation of cellulose as a substrate for hydrocarbon fuel production by Ascocoryne sarcoides (NRRL 50072)' submitted to the journal 'Journal of sustainable bioenergy systems' which is contained within this thesis.Ascocoryne sarcoides is an endophytic filamentous fungus isolated from Northern Patagonia that can produce petroleum-like fuel compounds directly from cellulose (Strobel et al., 2008). The aim of this project was to study volatile organic compound production by A. sarcoides. The project is different from many other cellulosic biofuel projects that explore alcohol production from pre-treated biomass fermentation. It focuses on the ability of A. sarcoides to convert cellulose and related simple sugars to fuel-related hydrocarbons. The influence of environmental conditions on the growth characteristics and compounds produced from normal metabolic processes was explored. Quantification of volatile production from growth on glucose showed the major compounds were ethanol and acetaldehyde, with the remainder of volatiles near 2 ppm from a continuously aerated culture. These volatiles included compounds of fuel-interest such as benzaldehyde, nonanal, 1-octen-3-ol and 1-butanol, 3-methyl-. Notable compounds with fuel related properties included isopentane, d-limonene, and cyclopropane, propyl-. These compounds all have octane ratings greater than 90 and enthalpies of combustion greater than 3200 kJ mol -1. Oxygen availability influenced the type and number of volatile compounds produced. For example, growth on a soluble cellulose substrate produced greater numbers of volatile compounds at oxygen limited concentrations and more alcohols, alkanes, aromatics, ketones, and esters were identified from mass spectrometry data. In addition, the oxygen availability influenced growth characteristics with a starting oxygen concentration of 7% was low enough to greatly inhibit growth. The growth substrate had a marked influence on the volatile compounds produced. A. sarcoides growth on microcrystalline cellulose produced a greater variety of hydrocarbon compounds compared to growth on glucose, and the highest yield of volatile organics was estimated at 105 mg (g biomass) -1 from the cellulose substrate. The fungus A. sarcoides demonstrated production of valuable fuel compounds on multiple carbon sources. Further work should carry on the analysis of culturing conditions and include evaluation of hydrocarbon yields on pre-treated cellulosic biomass. Studies of this nature should continue to advance knowledge of fungal biological potential for industrial processes.Item Lipid profiling, carbon partitioning, and inorganic carbon optimization to enhance growth and lipid accumulation in microalgae(Montana State University - Bozeman, College of Engineering, 2013) Lohman, Egan Jackson; Chairperson, Graduate Committee: Robin Gerlach; Robert D. Gardner, Luke Halverson, Richard E. Macur, Brent M. Peyton and Robin Gerlach were co-authors of the article, 'An efficient and scalable extraction and quantification method for algal derived biofuel' in the journal 'Journal of microbiological methods' which is contained within this thesis.; Robert D. Gardner, Luke Halverson, Brent M. Peyton and Robin Gerlach were co-authors of the article, 'Carbon partitioning in lipids synthesized by Chlamydomonas reinhardtii when cultured under three unique inorganic carbon regimes' submitted to the journal 'Applied phycology' which is contained within this thesis.; Robert D. Gardner, Todd Pedersen, Keith E. Cooksey, Brent M. Peyton and Robin Gerlach were co-authors of the article, 'An optimized inorganic carbon regime for enhanced growth and lipid accumulation in Chlorella vulgaris' submitted to the journal 'Biotechnology for biofuels' which is contained within this thesis.Microalgae are capable of accumulating high concentrations of lipids and other metabolites which can be used as precursor compounds for energy and valuable co-products. In order to fully exploit this resource, robust methods are needed to properly quantify and analyze the metabolites of interest. Additionally, understanding how and why these organisms synthesize these metabolites and developing optimized strategies for enhancing their metabolism is of paramount importance if algal biofuels and co-product development are to become commercially feasible. This dissertation represents the summary of work completed to develop analytical methods for quantifying lipid compounds synthesized by two Chlorophytes, Chlamydomonas reinhardtii sp. CC124 and Chlorella vulgaris UTEX 395, and the marine diatom Phaeodactylum tricornutum Pt-1. Additionally, C.reinhardtii was evaluated for factors that control and stimulate triacylglycerol (TAG) accumulation in microalgae by monitoring changes in lipid precursor compounds such as free fatty acids, mono- di- and tri-acylglycerides as well as fatty acids which were transesterified into fatty acid methyl ester (FAME); the bio-synthesized equivalent of diesel fuel. C. vulgaris was evaluated for optimized growth and lipid accumulation on various inorganic carbon substrates. This work resulted in a commercially applicable, two-phase growth/lipid accumulation regime which uses low grade sodium bicarbonate as the inorganic carbon substrate to enhance both growth and lipid accumulation and reduce the cost and resource overhead associated with using only carbon dioxide as the sole inorganic carbon source.Item Lipid production in algae stressed with sodium bicarbonate and sodium chloride(Montana State University - Bozeman, College of Engineering, 2013) Blaskovich, John Philip; Chairperson, Graduate Committee: Brent M. Peyton; Rob Gardner, Egan Lohman, Karen Moll, Luke Halverson, Robin Gerlach, Brent Peyton were co-authors of the article, 'The use of sodium bicarbonate and sodium chloride to stimulate lipid production in an algal isolate from Soap Lake, Washington' submitted to the journal 'Algal research' which is contained within this thesis.Microalgae may play an important role in the path to a more sustainable future by producing valuable hydrocarbons using inorganic carbon, sunlight, and non-food source competitive supplies of nitrogen and phosphorus. The prospect of growing microalgae for the production of a stable and dependable source of biofuel is plausible only if done at scale with intricate attention applied to the biochemistry, geochemistry, and environmental conditions of each system. Extreme environments with low proton activity and high salinity conditions may harbor microalgae suitable for large scale outdoor cultivation. Several algal isolates native to Soap Lake in Washington State were screened for biofuel potential and three isolates were selected for further studies. These three isolates were characterized to assess impacts on biofuel production studying high ionic strength in the form of sodium chloride (NaCl) in excess of 18g/L, and carbon supplemented treatments through the addition of inorganic carbon in the form of sodium bicarbonate (NaHCO 3). Further, the ability of NaHCO 3 and NaCl to trigger lipid production was determined. The study was centered on understanding differences between two factors that will likely have implications in large-scale algal raceway ponds: inorganic carbon limitation, speciation, or bioavailability, and evaporative conditions resulting in high concentrations of salt. In this study, cell concentration, cell dry weight, nitrate, pH, biofuel potential, extractable lipid potential, and DIC (dissolved inorganic carbon), were monitored over time. Isolate GK5La grown in standard medium had the highest concentration of cell dry weight at the end of the study. Cultures supplemented with sodium bicarbonate were determined to be the most efficient way to produce biofuel in the form of extractable lipids. Supplementation with sodium bicarbonate and spiking to a concentration of 18g/L sodium chloride showed to be the most productive way to make triacylglyceride (TAG). Fatty acid methyl ester (FAME) production on a concentration basis was greatest in the control treatment grown in standard medium.