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Item Sodium bicarbonate amendment for enhanced astaxanthin production from Haematococcus pluvialis(Montana State University - Bozeman, College of Engineering, 2019) Erturk, Berrak; Chairperson, Graduate Committee: Brent M. Peyton; Christian Lewis and Brent M. Peyton were co-authors of the article, 'Sodium bicarbonate amendment for enhanced astaxanthin production from Haematococcus pluvialis' submitted to the journal 'Algal research' which is contained within this thesis.Haematococcus pluvialis is a freshwater green microalga that is widely considered to be the richest natural source of the high value carotenoid astaxanthin. The use of bicarbonate salts as a means of efficiently delivering inorganic carbon in microalgal cultivation is a relatively new concept and its application is continuously growing. Previous studies have largely focused on increasing the lipid content in microalgae via the use of high concentrations of sodium bicarbonate under nitrogen deplete culture conditions. Lipid accumulation is directly related to astaxanthin production as astaxanthin is dissolved and stored in lipid bodies in H. pluvialis. Because of this relationship in H. pluvialis, the effects of sodium bicarbonate addition on astaxanthin production was investigated in this study. Due to its complex life cycle, H. pluvialis is commonly cultivated in two stages called the 'green' and 'red' stage. Different approaches have been proposed in each stage to increase the astaxanthin production, namely by growing microalgae under nutrient-limited conditions or resuspending the cells into nutrient deplete conditions. In this study, H. pluvialis (UTEX 2505) was cultivated in stirred (120 rpm) batch reactors containing MES-Volvox medium with a 12 h:12 h light/dark cycle. Sodium bicarbonate (2.5 mM) was used as an additional inorganic carbon source in the green stage and 50 mM of sodium bicarbonate was used as a trigger mechanism to induce astaxanthin production in the red stage. Following the trigger, the astaxanthin accumulation rate increased from 0.13 mg L ^-1 day ^-1 to 0.64 mg L ^-1 day ^-1 with an astaxanthin concentration of 1.56 + or - 0.01 mg L ^-1 and 3.95 + or - 1.25 mg L ^-1 respectively. Whereas, an addition of 2.5 mM sodium bicarbonate at the green stage increased the final astaxanthin accumulation rate up to 2.12 mg L ^-1 day ^-1 and the astaxanthin concentration to 11.2 + or - 0.56 mg L ^-1. Increasing biomass in the green stage resulted in higher astaxanthin content at the end of the red stage. In addition to increasing the total astaxanthin content, 2.5 mM of sodium bicarbonate led to faster nitrogen utilization during the green stage. With this faster utilization of nitrogen, the cultures were grown with a one-stage cultivation approach, where the astaxanthin production occurred in continuous mode.Item Organic amendments for enhancing microbial coalbed methane production(Montana State University - Bozeman, College of Engineering, 2017) Davis, Katherine Jean; Chairperson, Graduate Committee: Robin Gerlach; Robin Gerlach was a co-author of the article, 'Transition of biogenic coal-to-methane conversion from the laboratory to the field: a review of important parameters and studies' submitted to the journal 'International Journal of coal geology' which is contained within this thesis.; Shipeng Lu, Elliott P. Barnhart, Albert E. Parker, Matthew W. Fields and Robin Gerlach were co-authors of the article, 'Type and amount of organic amendments affect enhanced biogenic methane production from coal and microbial community structure' submitted to the journal 'Fuel' which is contained within this thesis.; Elliott P. Barnhart, Matthew W. Fields and Robin Gerlach were co-authors of the article, 'Fate of carbon during enhanced microbial methane production from coal with repeated organic amendment' submitted to the journal 'Energy & Fuels' which is contained within this thesis.; Matthew W. Fields and Robin Gerlach were co-authors of the article, '13C-labeled amendments for enhanced biogenic methane production in coal systems indicate increased coal-to-methane conversion' submitted to the journal 'Nature' which is contained within this thesis.; George A. Platt, Randy Hiebert, Robert Hyatt, Matthew W. Fields and Robin Gerlach were co-authors of the article, 'Development and pilot testing of column reactors for the study of anaerobic subsurface process' submitted to the journal 'International Journal of Coal Geology' which is contained within this thesis.Coalbed methane (CBM) is natural gas found in subsurface coal beds and supplies approximately 4-6% of the annual U.S. natural gas requirements. Many unmineable coal beds contain CBM produced by native microbial communities. Enhancing the microbial processes for coal-to-methane conversion can increase the rates of CBM production and the amount of extractable natural gas in these coal beds. Strategies for enhancing microbially-produced CBM must be logistically attainable and economically practical. The goal of this dissertation work was to determine a feasible methane enhancement strategy using organic amendments to increase microbial coal-to-methane conversion. Four organic amendments were tested in coal-containing batch microcosms. Increased coal-to-methane conversion was demonstrated with small amounts of amendment addition, and all four tested amendments increased methane production similarly. Subsequent amendment addition produced smaller amounts of additional methane which appeared to be primarily due to amendment-to-methane conversion. 13 C-labeled algal and yeast amendments were used in coal systems for tracking carbon for methane production. It was shown that <22% of the amendment carbon was converted to methane. By tracking amendment carbon, it became clear that carbon sources besides coal and amendment are utilized for methane production; these carbon sources potentially include organic and inorganic carbon in the formation water and inoculum. Amendment strategies tested in batch systems were scaled up and applied to column reactors. Methane production from coal increased with small amounts of 13 C-labeled algal amendment addition. However, unlike in batch experiments, methane production rates in the column flow reactors did not slow or cease after 60-90 days, and methane was still being produced after 176 days when the study was terminated.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 Characterization of surface colonization by microalgae using Botryococcus braunii and Dunaliella tertiolecta(Montana State University - Bozeman, College of Engineering, 2003) Jayawickramarajah, NarendrenItem 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.Item Control of triacylglycerol accumulation and bicarbonate-induced accumulation in microalgae(Montana State University - Bozeman, College of Engineering, 2012) Gardner, Robert David; Co-chairpersons, Graduate Committee: Brent M. Peyton and Keith Cooksey; Patrizia Peters, Brent M. Peyton, and Keith E. Cooksey were co-authors of the article, 'Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta' in the journal 'Journal of applied phycology' which is contained within this thesis.; Robert D. Gardner, Keith E. Cooksey, Florence Mus, Richard Macur, Karen Moll, Everett Eustance, Ross P. Carlson, Robin Gerlach, Matthew W. Fields, and Brent M. Peyton were co-authors of the article, 'Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte scenedesmus sp. and the diatom phaeodactylum tricornutum' in the journal 'Journal of applied phycology' which is contained within this thesis.; Egan Lohman, Robin Gerlach, Keith E. Cooksey and Brent M. Peyton were co-authors of the article, 'Comparison of CO 2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii' in the journal 'Biotechnology and bioengineering' which is contained within this thesis.Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated since these compounds can be used as precursors for food and fuel. This dissertation represents the summary of work completed to evaluate factors that control and stimulate triacylglycerol (TAG) accumulation in microalgae, and demonstrate the use of sodium bicarbonate as a chemical additive to induce TAG accumulation. Two Chlorophytes, Scenedesmus sp. WC-1 and Coelastrella sp. PC-3, were analyzed in pH buffered systems to determine the effect of pH on growth and TAG accumulation, both with and without nitrate deplete conditions. Medium nitrate and pH were found to be independent stress mechanisms and the causal effects of each were determined. Growth and TAG accumulation was optimized on the Chlorophyte Scenedesmus sp. WC-1 and the addition of bicarbonate was shown to arrest cellular replication and induce an elevated TAG accumulation rate. Thus, decreasing the total culturing time required for TAG production by half. To ascertain whether the addition of bicarbonate would give similar results in other algae, the marine diatom Phaeodactylum tricornutum Pt-1 and the model Chlorophyte Chlamydomonas reinhardtii were evaluated similar to WC-1. C. reinhardtii showed a cessation of the cell cycle with bicarbonate addition while P. tricornutum Pt-1 did not. However, the addition of bicarbonate was shown to induce lipid accumulation in both organisms. This demonstrates the broad use of bicarbonate as a means of inducing TAG accumulation in microalgae (Chlorophytes and diatoms, freshwater or marine).