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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 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 Biofuel potential, nitrogen utilization, and growth rates of two green algae isolated from a wastewater treatment facility(Montana State University - Bozeman, College of Engineering, 2011) Eustance, Everett O'Brien; Chairperson, Graduate Committee: Brent M. Peyton; Robin Gerlach (co-chair)Nitrogen removal from wastewater by algae provides the additional benefit of producing lipids for biofuel and biomass for anaerobic digestion. As ammonium is the renewable form of nitrogen produced during anaerobic digestion and one of the main nitrogen sources associated with wastewater, experiments focused on the optimization of growth and lipid production when grown on ammonium were evaluated. Scenedesmus sp. 131 and Kirchneriella sp. 92 were grown in a 14:10 light/dark cycle on ammonium, nitrate or urea in the presence of 5% CO 2 and ammonium and nitrate in the presence of air. Growth on nitrate and urea showed similar growth rates, and provided knowledge on the target growth rate for optimizing growth on ammonium. Results showed the pH decreased during exponential growth on ammonium in both 5% CO 2 and air, causing chlorophyll degradation. Growth on nitrate and air increased the pH of the medium and produced an increase in Nile Red fluorescence and biofuel potential for strain 131, but not for strain 92. Biological buffers were implemented to counteract the change in pH to prevent growth inhibition. Cultures were grown on 5% CO 2 or air, which showed that increased levels of CO 2 are required for increased growth, biofuel potential, and ammonium utilization. This increased the growth rates from 0.26 d -¹ to 1.04 d -¹ for strain 131 and 0.45 d -¹ to 1.31 d -¹ for strain 92. pH-controllers using 0.1 M KOH were used in experiments with 5% CO 2 with the understanding that buffers are limited to lab scale experiments and pH control would bridge the gap to industrial processes. The growth rate while utilizing pH-controllers showed similar growth rates to buffered experiments. Growth on nitrate, urea, and buffered ammonium with 5% CO 2 showed an increase in the biofuel potential for strain 92 in comparison to growth with air. Strain 131 had a decrease in biofuel potential when grown on ammonium compared to growth on nitrate or urea. Both strains showed increased levels of CO 2 is required to increase biofuel productivity.Item Algal biofilms, microbial fuel cells, and implementation of state-of-the-art research into chemical and biological engineering laboratories(Montana State University - Bozeman, College of Engineering, 2010) Menicucci, Joseph Anthony Jr.; Chairperson, Graduate Committee: Ron LarsenAlternative energy technologies become more attractive as the price of energy from fossil fuels becomes more expensive and the environmental concerns from their use mount. While a number of biological alternative energy technologies currently exist, a complete understanding of these technologies has yet to be developed. This dissertation characterizes an aspect of biological alternative energy technologies: the production of algal biofuels and energy conversion in microbial fuel cells. Specifically, this dissertation addresses the characterization of microalgae as a biofilm and the characterization of the power limitations of microbial fuel cells. The attachment and detachment of algae were observed using temporal microscopic imaging in a flow-cell with autofluorescence and staining techniques as part of a collaborative Montana State University and Idaho National Laboratory project. Colonies of algae exhibit many characteristics seen in bacterial biofilms: adherence; detachment and sloughing; difference in structure of an attached colony; varying strength of attachment on different surfaces; association of other organisms in an EPS matrix; and the heterogeneous nature of attached colonies. The characterization of a microbial fuel cell was completed in less than 30 minutes using an empirical procedure to predict the maximum sustainable power that can be generated by a microbial fuel cell over a short period of time. In this procedure, the external resistance was changed incrementally, in steps of 500 ohms every 60 seconds, and the anode potential, the cathode potential, and the cell current were measured. This procedure highlights the inherent limitations of energy conversion in a microbial fuel cell. A voltage/current characterization of the microbial fuel was also completed from the data collected. This dissertation also includes the evaluation of A Hands-On Introduction to Microbial Fuel Cells, a laboratory developed for an introductory chemical and biological engineering course. The experiment has been updated to include a voltage/current characterization of the microbial fuel cell. Learning objectives have been identified and pre- and post-laboratory activities have been developed for further implementation into a chemical and biological engineering curriculum.