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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 Genomic composition of green algae grown in high alkaline conditions(Montana State University - Bozeman, College of Agriculture, 2023) Goemann, Calvin Lee Cicha; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.Algae are responsible for 50% of global oxygen production and sequestration of CO 2 from the atmosphere. Algal photosynthesis plays a critical role in all aquatic ecosystems converting sunlight and CO2 into usable biomass. Algal growth and biomass production can be coopted to produce industrially relevant bioproducts like triacylglycerol (TAGs) that can be converted into biodiesel and provide a sustainable carbon-neutral alternative to fossil fuels. In high-stress environments, algae produce high levels of TAGs. Multiple stresses including nitrogen limitation and high pH impact algae physiology, but little is known about how algae shift their metabolism to produce TAGs in response to these stresses. This topic remains relatively unexplored due to the limited availability of complete algae genomes. Here we sequence and annotate the complete telomere-to-telomere genome of an alkali-tolerant green algae Chlorella sp. SLA-04. Genomic analysis supports a reclassification of Chlorophyta green algae and illuminates how SLA-04 adapts to diverse environmental conditions. Additionally, transcriptomic analysis revealed how Chlorella sp. SLA-04 rewires carbon metabolism in high alkaline and nutrient-deplete conditions to produce TAGs while minimizing photosynthetic oxidative stress. Together, we double the amount of publicly available telomere-to-telomere green algal genomes and use this resource to explore how algae respond to diverse environmental conditions in their native and industrial settings.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 Investigation of field relevant parameters for microbially enhanced coalbed methane scale up(Montana State University - Bozeman, College of Engineering, 2019) Platt, George Addison; Chairperson, Graduate Committee: Robin Gerlach; K. J. Davis, E. P. Barnhart, M. W. Fields and R. Gerlach were co-authors of the article, 'Optimization of 13C-algae amendment concentration for enhanced coal dependent methanogenesis' submitted to the journal 'International journal of coal geology' which is contained within this thesis.; K. J. Davis, H. D. Schweitzer, H. J. Smith, E. P. Barnhart, M. W. Fields, R. Gerlach were co-authors of the article, 'Algal amendment enhances biogenic methane production from coals of different thermal maturity' submitted to the journal 'International journal of coal geology' which is contained within this thesis.Energy production from coal is projected to decline significantly over the next 30 years, due to concerns over anthropogenic carbon emissions, climate change, and cost. As coal-based energy production decreases, the demand for natural gas is expected to increase. Coalbed methane (CBM), a biogenic natural gas resource found in subsurface coal beds, may aid in meeting the projected increase in demand. However, costs associated with traditional CBM extraction currently make utilizing this resource economically prohibitive due to slow coal-to-methane conversion rates and the necessity to treat co-produced water. Algae can be cultivated in co-produced formation water and the addition of very small amounts of this algal biomass can increase coal-to-methane conversion rates. The goal of this work was to determine the optimal algae amendment concentration for the enhancement of microbial coal-to-methane conversion to maximize return on investment. Concentrations of 13C-labeled algae amendment ranging from 0.01-0.50 g/L (equivalent to 0.0001-0.005 g per g of coal) were tested in coal-containing batch microcosms. Enhanced methane production was observed in all amended microcosms and maximum methane production occurred between 169-203 days earlier than in unamended microcosms. When as little as 0.01 g/L algae amendment was added, 13CH 4 and 12CH 4 tracking revealed that the improvement in coal-to-methane conversion kinetics was due to enhanced coal degradation. Increasing amendment concentrations to 0.05-0.50 g/L improved coal-to-methane conversion rates further, but improvements from amendment concentrations above 0.05 g/L were insignificant. The geologic scope of this CBM enhancement strategy was investigated by studying methane production from five coals ranging in thermal maturity. Biogenic methane was produced from all coals, with subbituminous coals generally producing more methane than thermally mature bituminous coals. The addition of algae amendment to thermally mature coal microcosms resulted in methane production that was comparable to production from unamended, thermally immature coals. This improvement was associated with an increased relative abundance of coal degrading microorganisms. Collectively, this work demonstrates that algae amendment concentrations can be reduced further (to 0.01-0.05 g/L) relative to the previously investigated concentrations (ranging from 0.1-0.5 g/L) and still improve coal-to-methane conversion rates for a range of coal sources.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 Evaluation of a green alga isolate for growth and lipid accumulation in coal bed methane water from the Powder River Basin(Montana State University - Bozeman, College of Engineering, 2015) Hodgskiss, Logan Henry; Chairperson, Graduate Committee: Alfred B. Cunningham; Matthew W. Fields was a co-author of the article, 'Growth of a native algal species in coal bed methane water for biofuel and biomass accumulation' submitted to the journal 'Environmental Science and Technology' which is contained within this thesis.Coal bed methane (CBM) production ponds are being constructed more frequently in areas such as the Powder River Basin in Montana and Wyoming where methane production has been active in the past decade. These ponds are currently not being utilized and are holding billions of gallons of water. The extracted water in these ponds is presently being discharged to local stream drainages or infiltrating into the surrounding soil. The environmental impacts of this increase in water can have negative effects on the surrounding areas. The purpose of this thesis is to explore the possibility of using CBM production ponds in the Powder River Basin, in Montana and Wyoming, for the growth of microalgae and the production of biodiesel from their accumulated lipids. Microalgae have been known to grow in other bodies of undesirable water and research has been ongoing on how to effectively use microalgae as a resource by stimulating lipid accumulation through the use of various environmental stressors. Coal bed methane ponds already provide a source of non-potable water for microalgae cultivation. Exploring the possibility of making these ponds a growth medium for microalgae is the first step in determining whether they can be turned into a productive energy resource. A native green alga, CBMW, has been isolated from a CBM production pond in northeastern Wyoming. CBMW has been cultured and grown under laboratory conditions in sterile CBM water and Bold's Basal Medium (BBM). Chlorophyll levels, biomass growth, pH, lipid accumulation, and water chemistry were tracked while CBMW was grown in sterile CBM water to understand how the alga responds to varying environmental conditions. When grown under the right environmental conditions isolate CBMW increased biomass and accumulated lipids. These results suggest that attempting to grow CBMW on a larger scale in CBM production water could be an effective method to produce biodiesel while utilizing a potentially problematic water source.Item The algal flora of the East Gallatin River as a contribution to the Algae of Montana(Montana State University - Bozeman, College of Agriculture, 1950) Jacobs, VedaItem A study of the East Gallatin River, Montana, using an algal bioassay (batch method) and some problems encountered(Montana State University - Bozeman, College of Agriculture, 1971) Griffin, Daniel PatrickItem Ecology of the diatom communities of the upper Madison River system, Yellowstone National Park(Montana State University - Bozeman, College of Agriculture, 1966) Roeder, Theodore ScottItem Influence of inorganic and organic nutrient enrichment on blue-green algal activity and relative biomass in a eutrophic southwest Montana reservoir(Montana State University - Bozeman, College of Letters & Science, 1991) Miller, Thomas David