Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Understanding physiological adaptations, metabolic potential and ecology in a novel photoautotrophic alga for biofuel production(Montana State University - Bozeman, College of Letters & Science, 2019) Corredor Arias, Luisa Fernanda; Chairperson, Graduate Committee: Matthew Fields; Elliot B. Barnhart, Al Parker, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Impact of temperature, nitrate concentration, PH and bicarbonate addition on biomass and lipid accumulation of a sporulating green alga' which is contained within this dissertation.; Thiru Ramaraj, Huyen Bui, Mensur Dlakic, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Genomic insights into a sporulating, non-motile, oligotrophic green microalga (PW95)' which is contained within this dissertation.; Huyen Bui, Thiru Ramaraj, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Transcriptomic profiling of Chlamydomonas-like PW95 cultivated in coal bed methane production water with the native microbial community' which is contained within this dissertation.; Anna J. Zelaya, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Associations between sympatric bacterial groups and a novel green alga cultivated in coal bed methane production water' which is contained within this dissertation.Commercial implementation of microalgal biomass as bio-oil/chemical feedstocks has been difficult to achieve, and challenges include water/nutrient sources, CO 2 delivery, and community dynamics of mixed cultures. We employed an integrated approach to the study of microalgal production systems to advance towards sustainable implementation of industrial microalgal biofuel production using a native alga (Chlamydomonas-like alga, PW95) isolated from Coal Bed Methane (CBM) production water. Our approach was based on the evaluation of PW95 physiological responses to combinations of growth constraints, the determination of its genomic and functional potential, phylogenetic relations and the implementation of an ecosystem view to algal biomass production. PW95 growth and lipid accumulation (biofuel potential) were ascertained in standardized media and CBM water through the evaluation of mixed effects of temperatures, nitrate levels, pH, and bicarbonate to elucidate interactions between multiple environmental variables and nutritional levels. The biofuel potential of PW95 ranges between 20-32% depending on culture conditions and our results suggest an important interaction between low nitrate levels, high temperature, and elevated pH for trade-offs between biomass and lipid production in the alga. Whole genome sequence was employed to predict biological and metabolic capacity in PW95, and the expression of these capabilities during growth in CBM water with the native microbial consortia was evaluated using RNA sequencing. genome determination and assembly resulted in a draft genome size of 92 Mbp with 14,000 genes predicted and 402 pathways mapped in the KEGG database. The gene complement of PW95 provided a glance into life in an oligotrophic environment (CBM water) and evidence of essential metabolic pathways for cell growth, survival and maintenance, also relevant for cultivation and value-added products generation. Microbial composition and shifts during growth were identified, as well as the algal phycosome. During growth in CBM water, PW95 appeared to be supported by a native microbial consortium and differential expression analysis showed basic metabolic functions and adaptive physiological responses. Our findings build on previous knowledge for improved algal culturing for biomass and industry-valued products while exploring the biology of an organism with relevant impact in energy and water resource management.Item The microbial community ecology of various systems for the cultivation of algal biodiesel(Montana State University - Bozeman, College of Letters & Science, 2017) Bell, Tisza Ann Szeremy; Chairperson, Graduate Committee: Matthew Fields; Bharath Prithiviraj, Brad D. Wahlen, Matthew W. Fields and Brent M. Peyton were co-authors of the article, 'A lipid-accumulating alga maintains growth in outdoor, alkaliphilic raceway pond with mixed microbial communities' submitted to the journal 'Frontiers in microbiology' which is contained within this thesis.; Emel Sen-Kilic, Tamas Felfoldi, Gabor Vasas, Matthew W. Fields and Brent M. Peyton were co-authors of the article, 'Bacteria and eukarya community during eutrophication and toxic cyanobacterial blooms in the alkaline Lake Velence, Hungary' which is contained within this thesis.; Lakotah Doig, Brent M. Peyton, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Characterizing the microbial community and its intrinsic ability to produce algal biodiesel in wastewater treatment lagoons' which is contained within this thesis.Algal based biofuel has the potential to aid in offsetting future fossil fuel consumption and demand, and lowering CO 2 emissions. Cultivation strategies are a pivotal component of achieving high biomass yield. Open outdoor pond systems are currently the most economically viable method for large-scale algae cultivation due to less energy for maintenance than closed systems. However, open pond cultivation is subject to microbial colonization, sometimes negatively impacting the algal crop. Thus, large-scale production is hindered by gaps in our fundamental understanding of microbial interactions and ecology. The following research aims to explore the interplay between cultivation methods, nutrient availability, community composition, lipid metabolism, and system ecology and identify cost effective concepts for algal lipid production. Using alkalinity to limit microbial colonization of an open system is investigated in Chapter 2 in which a monoculture of Chlorella vulgaris was successfully cultivated. A putative relationship with a Pseudomonas sp. was identified in which the exchange of key metabolites could have enhanced algal growth and limited contamination. Such interactions may minimize the need for pesticides and fertilizer subsequently reducing pollution and operating costs. Findings suggested that potentially beneficial algal-bacterial relationships occurring in alkaline conditions supported a productive and stable monoculture. Alkalinity, in addition to nutrient abundance, is further explored in a natural freshwater terminal lake system, presented in Chapter 3. Lake eutrophication coupled with temperature increases led to a toxic cyanobacterial bloom that reduced overall eukaryotic diversity. Insight gained on the interplay between alkalinity, nutrients, and community dynamics from this natural system was then applied to a series of artificial wastewater lagoons Chapter 4. Elevated lipid (g/L) was observed in this system partially facilitated by increased water residence time in the lagoons and elevated nitrogen availability. Differing alga community composition were observed during periods of elevated lipid in addition to higher biomass (cells/mL) suggesting that higher lipid volumes were the result of high biomass concentration and not necessarily the lipid productivity of specific alga taxa. The research presented utilizes traditional ecologic concepts like diversity and contributes to a more comprehensive understanding of community interactions helping to minimize cost, reduce pollution, and ultimately contribute to the realization of viable biodiesel.Item Diatom biofuels : optimizing nutrient requirements for growth and lipid accumulation in YNP isolate RGd-1(Montana State University - Bozeman, College of Letters & Science, 2012) Moll, Karen Margaret; Chairperson, Graduate Committee: Brent M. PeytonThe world's crude oil supply is decreasing at an alarming rate and no longer represents a long-term solution to meet energy needs. Development of renewable energy sources is required to meet transport fuel demands. Algal biofuels represent a potentially viable option. Diatom strain, RGd-1, isolated from Yellowstone National Park, produces high concentrations of lipids that can be used for biodiesel production. To increase cell numbers, RGd-1 was grown in six silica concentrations: without added silica, four silica concentrations within the soluble range (0.5-2mM), and one just above the soluble range (2.5 mM). Increasing the silica concentration resulted in an increase in total cell numbers and dry cell weight (DCW) with R ²=0.965. Silica depletion was verified by inductively coupled plasma mass spectrometry (ICP-MS). When grown in higher silica concentrations the medium reached a higher pH, which remained elevated. Nile Red fluorescence can be used as measurement of triacylglycerol (TAG). Once silica was depleted, Nile Red fluorescence increased. Unlike green algae and other diatoms, nitrate was never depleted when using the standard Bolds Basal Medium concentration (2.94 mM). RGd-1 never depleted nitrate from the growth medium and utilized only 1/3 of the original nitrate concentration (1 mM) by the time cells reached stationary phase. Therefore, the nitrate concentration was decreased to 1mM to induce a dual nitrate and silica stress. To increase the lipid content further, sodium bicarbonate was added to cells grown with each nitrate concentration (2.94 and 1 mM NO ₃-). Coupling nitrate limitation with sodium bicarbonate addition resulted in higher Nile Red fluorescence. RGd-1 fatty acids were primarily observed as C16:0, C16:1, C18:1-3 and C20:5, averaging at approximately 35, 30, 16 and 10%, respectively of the total lipid content. With exception of cells grown without added silica, the percent lipid content was approximately the same (30-40% (w/w) TAG (Triacylglycerol) and 70-80% (w/w) fatty acid methyl ester (FAME) grown under all conditions within the soluble range. However, when factoring in the dry cell weight from each system, it was observed that the TAG and FAME yields increased with silica concentration when normalized to DCW.Item Microbial conversion of biodiesel by-products to biofuel(Montana State University - Bozeman, College of Letters & Science, 2010) O'Shea, Kelly Frances; Chairperson, Graduate Committee: Matthew FieldsBiodiesel is an alternative liquid transportation fuel derived from biological oils. It is a renewable form of transportation fuel that can be easily integrated into society's current infrastructure. Biodiesel is cleaner burning than petroleum, emitting less carbon pollution and harmful toxins (i.e. sulfur, benzene). One of the major by-products from biodiesel production is crude glycerin. With the increased production of biodiesel, glycerin production will continue to increase. Glycerin was once considered a valuable co-product but now is considered a low-value by-product. In the following study, different co/tricultures of sulfate reducing bacteria (SRB) and methanogens were grown with crude glycerin as a means to convert the waste product into a renewable energy source, methane. The SRBs, Desulfovibrio vietnamensis and Desulfovibrio alcoholovorans 6133, were grown syntrophically, in different co/triculture combinations, with Methanococcus maripaludis, Methanoculleus marisnigri, and Methanosarcina acetivorans. Co/tricultures were investigated for the ability to produce methane via the utilization of pure glycerol, fractionated glycerin, and crude glycerin as carbon and energy sources. In order to gain insight into cellular physiology, glycerol, acetate, free fatty acid, and methane concentrations were measured throughout growth. The co/tricultures grew fastest on pure glycerol and experienced a lag phase in growth on fractionated glycerin and longer lag phases when transferred to crude glycerin. However, methane yields were similar on all three carbon sources. Methane production depended on the carbon source and culture composition. Co/tricultures growing on pure glycerol and fractionated glycerin displayed a decrease of methane production as growth rate increased. The opposite was seen with growth on crude glycerin. With most cultures, the addition of M. acetivorans increased methane concentrations significantly. M. acetivorans displayed the capability of utilizing the by-product, acetate, from SRB oxidation of glycerol and the methanol layer from fractionated and crude glycerin. M. acetivorans appeared to interfere with the coculturing of D. vietnamensis and M. marisnigri based on decreased methane production. Cocultures with M. maripaludis grew poorly and produced little methane when grown on the supernatant of M. acetivorans. This is the first study to characterize the utilization of crude glycerin from biodiesel production by syntrophic cultures of SRB and methanogenic archaea.