Browsing by Author "Reardon, Kenneth, committee member"

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Open Access
A plastic total internal reflection-based photoluminescence device for enzymatic biosensors
(Colorado State University. Libraries, 2013) Thakkar, Ishan G., author; Lear, Kevin L., advisor; Reardon, Kenneth, committee member; Collins, George, committee member
Growing concerns for quality of water, food and beverages in developing and developed countries drive sizeable markets for mass-producible, low cost devices that can measure the concentration of contaminant chemicals in water, food, and beverages rapidly and accurately. Several fiber-optic enzymatic biosensors have been reported for these applications, but they exhibit very strong presence of scattered excitation light in the signal for sensing, requiring expensive thin-film filters, and their non-planar structure makes them challenging to mass-produce. Several other planar optical waveguide-based biosensors prove to be relatively costly and more fragile due to constituent materials and the techniques involved in their fabrication. So, a plastic total internal reflection (TIR)-based low cost, low scatter, field-portable device for enzymatic biosensors is fabricated and demonstrated. The design concept of the TIR-based photoluminescent enzymatic biosensor device is explained. An analysis of economical materials with appropriate optical and chemical properties is presented. PMMA and PDMS are found to be appropriate due to their high chemical resistance, low cost, high optical transmittance and low auto-fluorescence. The techniques and procedures used for device fabrication are discussed. The device incorporated a PMMA-based optical waveguide core and PDMS-based fluid cell with simple multi-mode fiber-optics using cost-effective fabrication techniques like molding and surface modification. Several techniques of robustly depositing photoluminescent dyes on PMMA core surface are discussed. A pH-sensitive fluorescent dye, fluoresceinamine, and an O2-sensitive phosphorescent dye, Ru(dpp) both are successfully deposited using Si-adhesive gel-based as well as HydroThane-based deposition methods. Two different types of pH-sensors using two different techniques of depositing fluoresceinamine are demonstrated. Also, the effect of concentration of fluoresceinamine-dye molecules on fluorescence intensity and scattered excitation light intensity is investigated. The fluorescence intensity to the scattered excitation light intensity ratio for dye deposition is found to increase with increase in concentration. However, both the absolute fluorescence intensity and absolute scatter intensity are found to decrease in different amounts with an increase in concentration. An enzymatic hydrogen peroxide (H2O2) sensor is made and demonstrated by depositing Ruthenium-based phosphorescent dye (Ru(dpp)3) and catalase-enzyme on the surface of the waveguide core. The O2-sensitive phosphorescence of Ru(dpp)3 is used as a transduction signal and the catalase-enzyme is used as a bio-component for sensing. The H2O2 sensor exhibits a phosphorescence signal to scattered excitation light ratio of 100±18 without filtering. The unfiltered device demonstrates a detection limit of (2.20±0.6) µM with the linear range from 200µM to 20mM. An enzymatic lactose sensor is designed and characterized using Si-adhesive gel based Ru(dpp)3 deposition and oxidase enzyme. The lactose sensor exhibits the linear range of up to 0.8mM, which is too small for its application in industrial process control. So, a flow cell-based sensor device with a fluid reservoir is proposed and fabricated to increase the linear range of the sensor. Also, a multi-channel pH-sensor device with four channels is designed and fabricated for simultaneous sensing of multiple analytes.
Open Access
Algae-to-fuel pathways: integration of cultivation studies, process modeling, techno-economic analyses, and life cycle assessments
(Colorado State University. Libraries, 2022) Chen, Peter H., author; Quinn, Jason C., advisor; Bradley, Thomas, committee member; Marchese, Anthony, committee member; Reardon, Kenneth, committee member
Researchers have recognized the potential of microalgae for renewable fuels for several decades, with a sharp increase in interest in the past decade. Though progress in algal cultivation and conversion has been substantial, commercialization of algal fuels has not yet been achieved. Economic metrics must be balanced with renewable fuel goals such that algal fuels can be competitive with conventional petroleum fuels. Through process modeling, techno-economic analysis (TEA), and life cycle assessment (LCA), the work in this dissertation seeks to illuminate improvements to algal fuel systems and outline the steps required to advance algal fuels toward commercialization. This work heavily focuses on hydrothermal liquefaction (HTL), a thermochemical process that converts whole wet biomass into biocrude, a petroleum crude oil analog. An aqueous phase, a gaseous phase, and a solid phase are created alongside the primary biocrude product. The aqueous phase of HTL notably contains a high content of nitrogen, which could potentially be recycled back to algae cultivation. At a scale where algal biofuels would meet a significant portion of transportation fuel needs, the demand for nutrients, specifically nitrogen and phosphorus, would exceed current global agricultural production. While recycling the aqueous phase could alleviate the demand for fresh nutrients in algae cultivation, it also contains toxic components, which include heterocyclic nitrogen compounds and phenolic compounds. The first phase of this research is an experimental component that focuses on methods for improving the recyclability of nutrients in the aqueous phase. A novel use of adsorbents (activated carbon and ion-exchange resins) was discovered for reducing the presence of components that are toxic to algae growth. The second research phase is a comprehensive modeling effort of the HTL process. A process model was developed in Aspen Plus from a robust assessment of current literature. These results are fed into TEA and LCA models to fully demonstrate the effects that process uncertainties have on the viability of HTL. For example, the high-temperature conditions that define HTL require the material to maintain a subcritical liquid state, which complicates the assessment of accurate thermochemical properties due to the required pressure. To clarify this issue, the work in this research phase compares the estimated performance of algal HTL between different thermodynamic models. HTL environmental metrics beyond global warming potential and net energy ratio are also discussed for the first time. Uncertainties in conversion performance are bounded through a scenario analysis that manipulates parameters such as product yield and nutrient recycle (as discussed in the first research phase) to establish a range of economic results and environmental impacts. The work is supplemented with a publicly available model to support future hydrothermal liquefaction assessments and accelerate the development of commercial-scale systems. The third and final research phase compares HTL with a fractionation train called Combined Algal Processing (CAP) and takes into consideration the possibility of integrating HTL downstream of CAP. CAP can be described as a pretreatment and fermentation step followed by a lipid extraction step to extract carbohydrates and lipids, respectively, for fuel products. However, CAP cannot convert proteins to fuels, making the process highly dependent on feed composition from the cultivation stage. HTL's advantage over CAP is its relative agnosticism to composition, but it requires greater capital costs and is more energetically intensive. A fuzzy logic approach is proposed to compare CAP and HTL process models through relevant performance metrics and to map algal feed conditions that lead to optimal algae-to-fuel pathways. Thresholds are set for fuzzy membership functions in relevant performance objectives: minimum fuel selling price (MFSP), global warming potential (GWP), and net energy ratio (NER). The membership functions yield "satisfaction scores" for each objective and factor into an overall satisfaction score. Individual and overall satisfaction scores for each pathway are mapped to the full range of feed compositions (proteins, carbohydrates, and lipids). A composition-based algal growth model was then implemented to perform an uncertainty analysis through Monte Carlo simulations. The impact on satisfaction scores from varying other key process model parameters, such as algae productivity, individual process yields, process operating parameters, and life cycle inventory uncertainty are highlighted in these select scenarios.
Open Access
Conversion of lipid biomass to liquid hydrocarbons via pericyclic decarboxylations of α,β- and β,γ-unsaturated fatty acids using polycyclic aromatic hydrocarbon (PAH) solvent systems
(Colorado State University. Libraries, 2014) Romanishan, Michelle, author; Crans, Debbie C., advisor; Henry, Charles S., committee member; Barisas, George, committee member; Van Orden, Alan, committee member; Reardon, Kenneth, committee member
Development of a new process for converting lipid biomass, containing α,β- and β,γ-unsaturated fatty acids, to liquid hydrocarbon fuels (LHF) of varying carbon number is described in this dissertation. The theme for LHF production at present revolves around utilizing a catalytic system that requires high temperatures and pressures as well as multiple processing steps. The cost attributed to these types of processes has been a hindrance in moving the economy towards a cost-effective renewable fuel. Investigating possible catalyst-free processing techniques has led to the discovery of a lower energy reaction that utilizes specific unsaturated fatty acids into a cheap, high boiling point solvent system that has the ability to produce pure alkenes as liquid hydrocarbon fuels when heated to reflux temperature of the fatty acid. This sustainable process has been proven to decarboxylate α,β- and β,γ-unsaturated fatty acids via a pericyclic rearrangement. Using a high boiling, polycyclic aromatic hydrocarbon (PAH) solvent, such as phenanthrene or pyrene, pure alkene products in high yields have been obtained from heating α,β- or β,γ-unsaturated fatty acids to a temperature no higher than reflux of the acid. The successful process development and subsequent conversion of lipid-like biomass will be discussed at length and confirmed by ¹H NMR and GC/MS.
Open Access
Day and night for cyanobacteria: systems and synthetic biology approaches to understanding and engineering Synechocystis sp. PCC 6803 under day/night light cycles
(Colorado State University. Libraries, 2018) Werner, Allison Jean Zimont, author; Peebles, Christie A. M., advisor; Reardon, Kenneth, committee member; Prasad, Ashok, committee member; Heuberger, Adam, committee member
Photosynthetic organisms—including plants, algae, and cyanobacteria—harness sunlight as an energy source to grow, utilizing atmospheric carbon dioxide in the process. This ability can be harnessed for the sustainable production of food, fuels, and chemicals, reducing demand for petrol-based products and overall greenhouse gas emissions. Photosynthetic success rests on the efficient and timely capture of sunlight. Natural day/night cycles subject these organisms to changing energy availability, presenting a fundamental question: How do phototrophs regulate metabolism to thrive under daily and dramatic changes in energy supply? This question has significant impact on the productivity of plants, algae, and cyanobacteria. Cyanobacteria have been extensively engineered for the production of biofuels, polymers, and valuable pigments under continuous-light (CL) laboratory conditions. However, industrial production requires outdoor cultivation under diurnal light/dark (LD) cycles, where yield improvements in engineered strains observed in CL are lost in LD cycles. The success of industrially-productive cyanobacteria biotechnology is limited by the lack of appropriate strain engineering tools and gap in knowledge of photosynthetic metabolism under daily day/night light cycles. The aim of this thesis is therefore to improve the feasibility of cyanobacteria biotechnology in industrially-relevant conditions by integrating aspects of diurnal LD cycles into genetic tools and by expanding the current knowledge of dynamic photosynthetic metabolism. The first part of this thesis presents novel genetic engineering tools which enable light-entrained gene expression under diurnal LD cycles. The tools developed here enable engineering of temporally controlled chemical production under diurnal LD cycles, which we hypothesize will improve yield in outdoor cultivation environments. The second part of this thesis presents time-course characterization of growth and metabolite abundance under realistic diurnal LD cycles. Previous work was limited to on/off patterns of low light and restricted to detecting few metabolites. To expand the realism of light profiles and metabolite scope, a photobioreactor was engineered to supply sinusoidal patterns and intensity of light (sinLD cycles), and a multi-platform mass spectrometry workflow was developed to enable semi-comprehensive metabolite detection. Cyanobacteria growth under realistic diurnal sinLD cycles is presented for the first time, to our knowledge. We observe a short lag phase at the onset of day, followed by cell mass increase during the early day, cell division during afternoon and evening, and slight mass loss overnight. Further, comprehensive metabolite abundance every 30-120 minutes across a 24-hour diurnal sinLD cycle is presented. Insoluble C6 carbohydrates displayed sharp oscillations at the day/night transition; insoluble C5 carbohydrates and glucosamine display these in addition to abundance 're-sets' at the night/day transition. Free amino acids and nucleic acids increase immediately upon transition to light during the lag phase, followed by gradual incorporation into protein during the mass accumulation phase. Metabolites involved in central metabolism did not oscillate to the same extent as other pathways. Accumulation of phosphoenolpyruvate but not pyruvate during the light phase suggests a potential bottleneck. Integration of the metabolomics data into genome-scale metabolic models to perform dynamic flux balance analysis could improve the method by which engineering targets are identified for production in outdoor conditions. Together, this thesis demonstrates the need for revision of the current approach to cyanobacteria strain engineering. More broadly, this work highlights the dynamic nature of photosynthetic metabolism and motivates future investigations into metabolic regulation and metabolic flux under realistic day/night cycles.
Open Access
Development of genetic parts for improved control of translation initiation in Synechocystis sp. PCC 6803 with an application in biofuel production
(Colorado State University. Libraries, 2021) Sebesta, Jacob, author; Peebles, Christie A. M., advisor; Peers, Graham, committee member; Prasad, Ashok, committee member; Reardon, Kenneth, committee member
Metabolic engineering is developing into a field that can change the way we produce a wide variety of valuable chemicals. Many chemicals are already produced in microbial cultures. Metabolic engineering enables us to modify organisms to produce metabolites they don't usually produce, assuming an enzyme can be identified in another organism that catalyzes the formation of that product (or an enzyme can be designed for that task through protein engineering). The distribution of accumulated metabolites can also be altered. There are some cases where metabolites can be accumulated through cultivation practices. Methods of metabolic engineering to overexpress, knockdown, or knockout native enzymes provide additional tools to alter cellular metabolism and drive accumulation of those products. Precise control over gene expression is central to these efforts. To avoid competition with human food crops and the resources need to produce them, cyanobacteria may be utilized for production of valuable chemicals. Through photosynthesis, they can utilize carbon dioxide from geological formations or from industrial waste streams. Since most metabolic engineering has been developed in E. coli and yeast, it was necessary to first adapt the basic methods for use in cyanobacteria. Along with my co-authors Dr. Allison Werner and Dr. Christie Peebles, we reviewed methods for producing genetically modified Synechocystis Sp. PCC6803 (S. 6803). To facilitate the generation of strains with many modifications, we covered the method developed in the Peebles Lab for making markerless selections which remove any antibiotic selection markers. A previous graduate student in the Peebles lab, Stevan Albers, found that strong promoter-ribosome binding site combinations that drove high expression of GFP did not necessarily result in high expression when used to drive expression of a different gene. Therefore, in our work to produce bisabolene in S. 6803 we tested many ribosome binding sites. In addition, we tested five different codon optimizations of the bisabolene synthase to ensure that expression was not prevented by slow translation elongation. We found that the simple measure of the codon adaptation index (CAI) correlated with expression of the five different codon optimizations. Using a thermodynamic model of translation initiation, we designed ten ribosome binding sites to increase bisabolene synthase expression by 10-fold. Only one of those designs actually approached a 10-fold increase, highlighting the need to continue testing several ribosome binding sites to achieve a desired expression level. Since industrial cultivation of cyanobacteria occurs outdoors, subject to natural light:dark cycles, we tested two of the designed strains in light:dark cycles. The strains reached similar bisabolene titers after being exposed to the same amount of total light period as those previously tested in continuous light. Overall, this work increased the highest bisabolene titer reported in cyanobacteria by approximately 10-fold. The need to test many ribosome binding sites limits progress in cyanobacterial metabolic engineering. The research of others suggest that ribosome binding sites interact with coding sequences by forming secondary structures with different free energy of folding. The estimation of the free energy of folding may be inaccurate, and, further, the kinetics of such folding may also be important to translation initiation rates. We tested two different designs to limit the impacts that secondary structures that span either side of the start codon may have on translation initiation rates in both E. coli and S. 6803. Utilization of a 21-nucleotide leader sequence after the start codon to make the sequence context consistent for ribosome binding sites between different coding sequences did not improve the correlation found between the expression of two different reporter genes in either organism. Bicistronic designs use translational coupling between an upstream open reading frame and the gene of interest with a ribosome binding site contained within the upstream open reading frame to re-initiate translation. This design exploits the helicase activity of ribosomes in elongation mode to actively unfold the secondary structure around the start codon of the gene of interest. We expected this activity to reduce the impacts of secondary structure and improve the correlation in expression between two different reporter genes. Intriguingly, the correlation was much improved in E. coli, but not in S. 6803. Together, this dissertation suggests that there are important differences in translation initiation between E. coli and S. 6803. Improved ribosome binding site design for cyanobacteria would facilitate further increases in terpenoid production both by enabling higher expression of heterologous terpenoid synthases and by reducing the number of strains that must be tested to achieve the desired expression level for each enzyme. Future directions suggested by this work include studies of translation initiation mechanisms in cyanobacteria, development of cell-free expression systems to facilitate rapid testing of many different genetic constructs, and further efforts at pathway engineering to increase terpenoid titer and productivity in cyanobacteria.
Open Access
Engineering stabilized enzymes via computational design and immobilization
(Colorado State University. Libraries, 2016) Johnson, Lucas B., author; Snow, Christopher, advisor; Reardon, Kenneth, committee member; Peebles, Christie, committee member; Peersen, Olve, committee member
The realm of biocatalysis has significantly matured beyond ancient fermentation techniques to accommodate the demand for modern day products. Enzymatically produced goods already influence our daily lives, from sweeteners and laundry detergent to blood pressure medication and antibiotics. Protein engineering has been a major driving force behind this biorevolution, yielding catalysts that can transform non-native substrates and withstand harsh industrial conditions. Although successful in many regards, computational design efforts are still limited by the crude approximations employed in searching a complex energy landscape. Advancements in protein engineering methods will be necessary to develop our understanding of biomolecules and accelerate the next generation of biotechnology applications. Our work employs a combination of computational design and simulation to achieve improved enzyme stability. In the first example, an enzyme used in the production of cellulosic biofuels was redesigned to remain active at high temperature. An initial approach involving consensus sequence analysis, predicted point mutation energy, and combinatorial optimization resulted in a sequence with reduced stability and activity. However, by using recombination methods and molecular dynamics simulations, we were able to identify specific mutations that had a stabilizing or destabilizing effect, and we successfully isolated mutations that benefited enzyme stability. Our iterative approach demonstrated how common design failures could be overcome by careful interpretation and suggested methods for improving future computational design efforts. In the second example, a cellulase was designed to have a high net charge via selected surface mutagenesis. “Supercharged” cellulases were experimentally characterized in various ionic liquids to assess the effect of high ion concentration on enzyme stability and activity. The designed enzymes also provided an opportunity to systematically probe the protein-solvent interface. Molecular dynamics simulations showed how ions influenced protein behavior by inducing minor unfolding events or by physically blocking the active site. Contradictory to previous reports, charged mutations only appeared to alter the affinity of anions and did not significantly change the binding of cations at the protein surface. Understanding the different modes of enzyme inactivation could motivate targeted design strategies for engineering protein resilience in ionic solvents. In addition to the discussed computational design methods, immobilization strategies were identified for capturing enzymes within porous protein crystals. Immobilization offers a generic approach for improving enzyme stability and activity. Our preliminary studies involving horseradish peroxidase and other enzymes suggested protein scaffolds could be employed as an effective immobilization material. Co-immobilizing multiple enzymes within the porous material led to improved product yield via exclusion of off-pathway reactions. Although future studies will be required to assess the potential capabilities of this immobilization strategy in comparison to other materials, preliminary results suggest protein crystals offer a favorable, controlled environment for immobilizing enzymes. The diversity of approaches presented in this thesis emphasizes that there are many options for engineering enzyme stability. Extending the lessons learned from our cellulase engineering to the greater field of rational protein design promotes the concept of biomolecules as designable entities. By establishing the shortcomings of our designs and suggesting routes for improvement, we anticipate our design methods and immobilization strategies will procure continued interest from the biotechnology community. The toolsets we developed for cellulases can be directly transferred to other enzymes and have the potential to impact a range of protein engineering applications.
Open Access
Environmental and economic evaluation of algal-based biofuels through geographically resolved process and sustainability modeling
(Colorado State University. Libraries, 2023) Quiroz, David, author; Quinn, Jason C., advisor; Windom, Bret, committee member; Willson, Bryan, committee member; Reardon, Kenneth, committee member
Advanced algal renewable fuels have been the subject of extensive research during the last decades. Their advantages over conventional biofuel feedstocks position algal biomass as a promising feedstock for the development of a sustainable and circular bioeconomy. Despite recent technological improvements, techno-economic analyses (TEAs) show that algae-derived fuels fail to be cost-competitive with petroleum fuels. Moreover, results from life-cycle assessments (LCAs) indicate declining greenhouse gas emissions when compared to petroleum fuels, but their water, health and air pollution impacts are still uncertain. This is explained by the fact that most published TEAs and LCAs of algal systems are not supported by high-resolution models and can only provide average sustainability metrics based on results from restricted data sources. These assessments often lack the resolution to correctly analyze the temporal and regional variations of biomass yields which have a direct impact on TEA and LCA metrics. Based on the current state of the field, there is a critical need to develop dynamic models that can inform sustainability assessments and consequently assist decision-making and technology development. This first part of this research work focuses on establishing the foundations for spatially explicit and temporally resolved LCA and TEA by developing and validating models that capture the thermal and biological dynamics of open algal cultivation systems. The modeling work is heavily focused on providing accurate predictions of evaporation losses in open algae raceway ponds and investigating the effects of evaporation rates on pond temperatures and growth rates. To date, this is the first modeling effort focused on predicting the evaporation losses of open algal ponds at the commercial scale. The outputs from the thermal model are then used to inform a biological algae growth model that is validated with experimental data representing the current biomass productivity potential. When integrated with hourly historical weather data, the modeling tools provide spatiotemporal mass and energy balances of the algal cultivation, dewatering, and conversion to fuel processes. These results are then leveraged with sustainability tools such as LCA and TEA to provide sustainability metrics at a high temporal and spatial scale. After developing a robust modeling framework, the modeling tool is leveraged with two distinct water LCA methods to provide a comprehensive assessment of the water impacts of algae-derived renewable diesel production across the United States. First, a water footprint analysis is conducted to understand the direct freshwater and rainwater consumption of algal cultivation and provide a framework for comparison to traditional biofuel feedstocks. The second method provides a county-level water scarcity footprint by analyzing the impact of algal systems on local water demand and availability. This assessment allows for the proper identification of potential algal sites for algal cultivation and locations where the deployment of algal systems will exacerbate local water stress. Ultimately, this research chapter provides the first holistic investigation of the water consumption and environmental water impacts of algal systems across the U.S. and establishes benchmarks for comparison to other fuels. Finally, the work comprising the third research chapter includes a novel global sustainability assessment that integrates the developed process modeling framework with regional-specific TEA and LCA. The spatially explicit TEA considers regional labor costs, construction factors, and tax rates to assess the economic viability of algal biofuels across 6,685 global locations. Similarly, a well-to-wheels LCA was performed by accounting for the regional life cycle impacts associated with electricity generation, hydrogen, and nutrient production across ten different environmental categories including health, air pollution, and climate impacts. This framework enables the identification of algal sites with optimal productivity potential, environmental impacts, and economic viability. Discussion focuses on the challenges and opportunities to reduce costs and environmental impacts of algal biofuels in various global regions.
Open Access
Evaluation of wastewater as a nutrient source for the cultivation of the model cyanobacterium Synechocystis sp. PCC6803
(Colorado State University. Libraries, 2017) Hughes, Alexander, author; Peers, Graham, advisor; Sloan, Daniel, committee member; Reardon, Kenneth, committee member
The rising demand for more sustainable and renewable energy sources has led to the development of using photosynthetic microalgae and cyanobacteria for biofuel feedstocks. Microalgae and cyanobacteria offer an attractive solution over the cellulosic and lignocellulosic feedstocks of first and second generation biofuels that compete for arable land, nutrients and water necessary for sufficient food crop production. It has been proposed for several decades that wastewater could be a sustainable and affordable source of water and nutrients. Phycoremediation of wastewater by microalgae as biofuel feedstocks could provide beneficial environmental health impacts by preventing eutrophication of fresh water supplies. Many eukaryotic microalgae have been grown in diluted and/or modified wastewaters. The growth of cyanobacteria on wastewaters has not been nearly as well characterized. Cyanobacteria grown on wastewaters could be an ideal feedstock for advanced biofuels, since cyanobacteria have a more extensively established molecular toolbox for genetic engineering. The first aim of this thesis was to evaluate wastewater centrate as a growth medium for the cultivation of the cyanobacterium Synechocystis sp. strain PCC6803 (Chapter 1). Centrate was collected from the Drake Water Reclamation Facility (Fort Collins, CO) and filter sterilized to allow axenic culturing of PCC6803 under controlled laboratory conditions. PCC6803 was grown in up to 21% filtered centrate diluted in sterile water; while higher concentrations completely inhibited growth. Nitrogen drawdown from centrate by PCC6803 was then analyzed. Surprisingly, the drawdown of nitrogen from the centrate media correlated poorly with the amount of cyanobacterial biomass. The cell densities of cultures grown in centrate were all significantly lower than that of PCC6803 grown in BG-11 indicating that diluted centrate does not provide adequate nutrients to support optimal growth. Abiotic precipitation of nitrogen was then determined to dominate the removal of nitrogen from the cultivation media. Furthermore, it suggested that centrate lacks a critical nutrient to support robust growth of PCC6803. The second aim of this thesis was to augment the nutrient composition of wastewater in order to optimize PCC6803 growth and nutrient removal (Chapter 2). A series of bioassays were performed to elucidate the limiting macronutrient in centrate. Adding 304 μM Na2SO4 – equivalent to the concentration of SO42in BG-11 media – yielded final cell densities that were only 4% lower than those observed in cultures grown in the synthetic, standard media (BG-11). Exogenous supplementation of Na2SO4 also improved total dissolved nitrogen (TN) drawdown for centrate grown PCC6803 cultures. In Na2SO4 amended centrate, PCC6803 was able to grow to significantly higher cell densities, permitting the removal of 69% of the TN in diluted centrate. Transcript abundance of the sulfate transporters encoded by the spbA-cysTWA operon were found to be upregulated when grown in centrate, confirming that PCC6803 experienced S-limitation during growth on this media. Hydrogen sulfide gas (H2S) is an undesirable product of the biological nutrient removal process due to its pungent odor. Currently, H2S produced at the DWRF is vented to biofilters consisting of wood chips and compost where sulfide oxidizing microbes convert sulfide into elemental sulfur. Therefore, endogenously sourced sulfur supplementation from H2S into centrate could provide a viable sulfur source to support the cultivation of PCC6803. We have shown that sulfur supplementation improves the phycoremediation of nutrients in centrate. Cultivation of PCC6803 on centrate supplemented with endogenously sourced sulfur provides an industrially feasible method for combining wastewater treatment with advanced biofuel production.
Open Access
From waste to energy: a techno-economic analysis and life cycle analysis of liquid biochemical production from wet wastes through enhanced anaerobic digestion
(Colorado State University. Libraries, 2022) Soliman, Abdallah, author; Quinn, Jason C., advisor; Reardon, Kenneth, committee member; Windom, Bret, committee member
Wet wastes such as manure and food wastes present problems due to disposal costs and environmental impacts. Low value products and methane leaks limit the sustainability and viability of current anaerobic digestion for treatment of wet waste. Electrochemically enhanced conversion of wet wastes diverts carbon from low-value methane into volatile fatty acids that are subsequently upgraded to improve anaerobic digestion sustainability and generate biochemicals which are seamlessly compatible with the current infrastructure. A chain elongation pathway and a bioconversion pathway are used to produce caproic acid and n-butanol, respectively. Techno-economic analysis and life cycle assessment are used to demonstrate the economic and environmental viability of the technology. The economic analysis generates market competitive minimum selling prices of $1.05 per kg for the caproic acid pathway and $2.25 per kg for the n-butanol pathway. The baseline environmental analysis yields an environmentally unfavorable GWP of 72.1 g CO2-eq·MJcaproic acid-1 for the chain elongation pathway whereas the GWP of the bioconversion pathway (24.0 g CO2-eq·MJn-butanol-1) qualifies it as a renewable fuel under the RFS program. Using scenario and sensitivity analyses, critical research areas were highlighted to guide future work and improve the performance and sustainability of the technology.
Open Access
Genome-scale metabolic modeling of cyanbacteria: network structure, interactions, reconstruction and dynamics
(Colorado State University. Libraries, 2016) Joshi, Chintan Jagdishchandra, author; Prasad, Ashok, advisor; Peebles, Christie A. M., committee member; Reardon, Kenneth, committee member; Peers, Graham, committee member
Metabolic network modeling, a field of systems biology and bioengineering, enhances the quantitative predictive understanding of cellular metabolism and thereby assists in the development of model-guided metabolic engineering strategies. Metabolic models use genome-scale network reconstructions, and combine it with mathematical methods for quantitative prediction. Metabolic system reconstructions, contain information on genes, enzymes, reactions, and metabolites, and are converted into two types of networks: (i) gene-enzyme-reaction, and (ii) reaction-metabolite. The former details the links between the genes that are known to code for metabolic enzymes, and the reaction pathways that the enzymes participate in. The latter details the chemical transformation of metabolites, step by step, into biomass and energy. The latter network is transformed into a system of equations and simulated using different methods. Prominent among these are constraint-based methods, especially Flux Balance Analysis, which utilizes linear programming tools to predict intracellular fluxes of single cells. Over the past 25 years, metabolic network modeling has had a range of applications in the fields of model-driven discovery, prediction of cellular phenotypes, analysis of biological network properties, multi-species interactions, engineering of microbes for product synthesis, and studying evolutionary processes. This thesis is concerned with the development and application of metabolic network modeling to cyanobacteria as well as E. coli. Chapter 1 is a brief survey of the past, present, and future of constraint-based modeling using flux balance analysis in systems biology. It includes discussion of (i) formulation, (ii) assumption, (iii) variety, (iv) availability, and (v) future directions in the field of constraint based modeling. Chapter 2, explores the enzyme-reaction networks of metabolic reconstructions belonging to various organisms; and finds that the distribution of the number of reactions an enzyme participates in, i.e. the enzyme-reaction distribution, is surprisingly similar. The role of this distribution in the robustness of the organism is also explored. Chapter 3, applies flux balance analysis on models of E. coli, Synechocystis sp. PCC6803, and C. reinhardtii to understand epistatic interactions between metabolic genes and pathways. We show that epistatic interactions are dependent on the environmental conditions, i.e. carbon source, carbon/oxygen ratio in E. coli, and light intensity in Synechocystis sp. PCC6803 and C. reinhardtii. Cyanobacteria are photosynthetic organisms and have great potential for metabolic engineering to produce commercially important chemicals such as biofuels, pharmaceuticals, and nutraceuticals. Chapter 4 presents our new genome scale reconstruction of the model cyanobacterium, Synechocystis sp. PCC6803, called iCJ816. This reconstruction was analyzed and compared to experimental studies, and used for predicting the capacity of the organism for (i) carbon dioxide remediation, and (ii) production of intracellular chemical species. Chapter 5 uses our new model iCJ816 for dynamic analysis under diurnal growth simulations. We discuss predictions of different optimization schemes, and present a scheme that qualitatively matches observations.
Open Access
Geographical assessment of algal productivity and water intensity across the United States
(Colorado State University. Libraries, 2021) Quiroz, David, author; Quinn, Jason C., advisor; Marchese, Anthony, committee member; Reardon, Kenneth, committee member
Water consumption due to evaporation in open algal cultivation systems represents a significant research gap in the resource assessment literature. Existing algal evaporation models often lack high spatiotemporal resolution or are not validated with experimental systems. This study presents a geographical and temporal assessment of the water requirements for commercial-scale production of algae biomass through a dynamic integrated thermal and biological modeling framework. Water demands were calculated through a validated dynamic thermal model which predicts temperature with an accuracy of -0.96 ± 2.72 °C and evaporation losses with a 1.46 ± 5.92 % annual accuracy. The biological model was validated with experimental data representing the current state of technology and shows an average error of -4.59 ± 8.13 %. The integrated thermal growth model was then utilized to simulate the water demands for biomass production of a 400-hectare algae farm at 198 different locations across the United States over a period of 21 years. Simulation outputs were used to determine algal protein yields, based on protein content, and fuel production via hydrothermal liquefaction. This foundation is integrated with life cycle methodology to determine the water footprint of algal biomass, proteins, and biofuels and to compare them to those of traditional energy crops and conventional fuels. Results indicate that less water-intensive cultivation can be achieved in the Gulf Coast region, where the average water footprints of the three simulated pathways were determined to be 155 m3 water tonne-1 biomass, 371 m3 water tonne-1 algal proteins, and 11 m3 GJ-1 biofuel. The water footprints of algal systems were found to be more favorable when compared to traditional biomass feedstocks such as soybeans and corn. However, when compared to petroleum-based fuels, results emphasize the need for more water-efficient strategies to reduce the water demands of algal cultivation. This work also incorporates a novel temperature tolerance assessment to identify the geographically specific temperature limits for algal strains in a commercial-scale facility. Results highlight the importance of high temporal and spatial resolution when modeling culture temperature, evaporative loss, and algae growth rate.
Open Access
Geographically-resolved life cycle assessment and techno-economic analysis of engineered climate solutions with an innovative framework for decision support
(Colorado State University. Libraries, 2024) Greene, Jonah Michael, author; Quinn, Jason C., advisor; Reardon, Kenneth, committee member; Coburn, Tim, committee member; Baker, Daniel, committee member
The urgent challenge of addressing climate change requires a thorough evaluation of engineered solutions to ensure they are both economically viable and environmentally sustainable. This dissertation performs a comprehensive assessment of two key climate technologies: microalgae biorefineries for biofuel production and anaerobic digestion (AD) systems for reducing greenhouse gas (GHG) emissions on dairy farms. Using high-resolution life cycle assessment (LCA) and techno-economic analysis (TEA), it provides detailed insights into the sustainability performance of these technologies. In addition, this work goes further by introducing a decision-support framework that improves the interpretation of LCA and TEA results, enhancing decision-makers' ability to form sustainable policies and implement actionable outcomes that drive the transition to green energy solutions. The first segment of this dissertation integrates high-resolution thermal and biological modeling with LCA and TEA to evaluate and compare two different microalgae biorefinery configurations targeting renewable diesel (RD) and sustainable aviation fuel (SAF) production in the United States. A dynamic engineering process model captures mass and energy balances for biomass growth, storage, dewatering, and conversion with hourly resolution. These configurations support facilities in remote areas and cultivation on marginal lands, enabling large-scale biofuel production. The two pathways under examination share identical biomass production and harvesting assumptions but differ in their conversion processes. The first pathway evaluates hydrothermal liquefaction (HTL) to produce RD, while the second explores the Hydroprocessed Esters and Fatty Acids (HEFA) process to produce SAF. Results indicate that the Minimum Fuel Selling Price (MFSP) for RD could decrease from $3.70-$7.30 to $1.50-$4.10 per liter of gasoline equivalent, and for SAF from $9.90-$19.60 to $2.20-$7.30 per liter under future scenarios with increased lipid content and reduced CO2 delivery costs. Optimization analyses reveal pathways to achieve an MFSP of $0.75 per liter and 70% GHG emissions reductions compared to petroleum fuels for both pathways. Additional analysis covers the water footprint, land-use change emissions, and other environmental impacts, with a focus on strategic research and development investments to reduce production costs and environmental burdens from microalgae biofuels. Beyond renewable transportation fuels, achieving a sustainable energy future will require innovations in the circular economy, such as waste-to-energy systems that reduce GHG emissions while simultaneously producing renewable energy. Accordingly, the second segment of this dissertation examines the GHG reduction potential of adopting AD technology on large-scale dairy farms across the contiguous United States. Regional and national GHG reduction estimates were developed through a robust life cycle modeling framework paired with sensitivity and uncertainty analyses. Twenty dairy configurations were modeled to capture key differences in housing and manure management practices, applicable AD technologies, regional climates, storage cleanout schedules, and land application methods. Monte Carlo uncertainty bounds suggest that AD adoption could reduce GHG emissions from the large-scale dairy industry by 2.45-3.52 million metric tons (MMT) of CO2-equivalent (CO2-eq) per year when biogas is used solely in renewable natural gas programs, and as much as 4.53-6.46 MMT of CO2-eq per year when combined heat and power is implemented as an additional biogas use case. At the farm level, AD technology may reduce GHG emissions from manure management systems by 58.1-79.8%, depending on the region. The study highlights the regional variations in GHG emissions from manure management strategies, alongside the challenges and opportunities surrounding broader AD adoption. It is vital to confirm that engineered climate solutions offer real improvements and to identify key enhancements needed to replace existing technologies. This process hinges on effective policy and decision-making. To address these challenges, the final segment of this dissertation introduces the Environmental Comparison and Optimization Stakeholder Tool for Evaluating and Prioritizing Solutions (ECO-STEPS). ECO-STEPS offers a decision-support framework that utilizes outputs from LCA and TEA to help decision-makers evaluate and prioritize engineered climate solutions based on economic viability, environmental impacts, and resource use. The tool's framework combines stakeholder rankings for key sustainability criteria with diverse statistical weighting methods, offering decision support aligned with long-term sustainability goals across various technology sectors. Applied to a biofuels case study, ECO-STEPS compares algae-based RD, soybean biodiesel (BD), corn ethanol, and petroleum diesel, using an expert survey to determine criteria rankings. Results indicate that soybean BD is a strong near-term solution for the biofuels sector, given its economic viability and relatively low environmental impacts. In contrast, corn ethanol, while economically competitive, demonstrates poor environmental performance across multiple sustainability themes. Algae-based RD emerges as a promising long-term option as ongoing research and development reduce costs. The results of this case study illustrate that ECO-STEPS provides a flexible and comprehensive framework for stakeholders to navigate complex decision-making processes in the pursuit of sustainable climate solutions. In conclusion, the integration of high-resolution LCA, TEA, and a stakeholder-driven decision-support framework in this dissertation presents a comprehensive approach to evaluating engineered climate solutions. The results from these studies provide geographically resolved insights into the sustainability performance of key climate technologies, offering actionable pathways for optimizing biofuel production, reducing GHG emissions, and supporting sustainable decision-making to advance the transition to a green economy.
Open Access
Metabolic engineering of the cyanobacterium Synechocystis sp. PCC 6803 for the production of astaxanthin
(Colorado State University. Libraries, 2016) Albers, Stevan Craig, author; Peebles, Christie A. M., advisor; Reardon, Kenneth, committee member; Prasad, Ashok, committee member; Peers, Graham, committee member
Synechocystis sp. PCC 6803 is a photosynthetic eubacterium capable of using light energy to generate biomass from atmospheric CO2 and is considered to be the model organism of photosynthetic microbes. Much of the knowledge accumulation related to this organism has centered on the cellular photosynthetic process because this organism has many similarities to the chloroplasts of higher order plants. Synechocystis also shows great promise as a microbial cell factory, as scientific studies describing metabolite production from this organism continue to accumulate in the literature. While these studies highlight the considerable amount of gains made in regards to production in Synechocystis, they also shed light on the considerable amount of gaps in knowledge regarding many aspects of this organism. As the field of metabolic engineering continues to grow within Synechocystis, researchers must continue to develop production pathways that leverage comprehensive engineering strategies that help in shedding light on critical engineering hurdles. This information is critical for the successful development of photosynthetic microbes as cellular production platforms capable of generating titers similar to those seen in other cellular systems utilized to generate economically viable metabolites for humankind. In this work, we utilized several metabolic engineering strategies to manipulate the carotenoid biosynthesis pathway in Synechocystis for the production of the non-native carotenoids, astaxanthin as well as canthaxanthin. A Synechocystis mutant was engineered with an insertion of a β-carotene di-ketolase gene crtW148 from Nostoc punctiforme, insertion of an additional copy of the endogenous β-carotene hydroxylase gene crtR from Synechocystis, and an open reading frame disruption of the endogenous β-carotene mono-ketolase gene crtO. These manipulations generated a mutant capable of an increase in the overall carotenoid content by 178 ± 10% % of that seen in wild type cells as well as astaxanthin titers that reached production rates of 1.11 ± 0.07 mg/l/day and canthaxanthin titers reaching 1.49 ± 0.05 mg/l/day. To add upon this work, we leveraged several promoters, the PSCA6-2 promoter as well as the PsigA promoter to control the expression of the crtW148 gene within several constructs. These promoters were generated in a research study we performed that leveraged rational design strategies to develop a suite of promoters capable of driving gene expression as various strengths within Synechocystis. This study generated a library of 10 promoter-constructs capable of a dynamic range of expression strength, exhibiting a 78 fold change between the lowest expressing promoter, Psca8-2 and the highest expressing promoter, Psca3-2 when tested within Synechocystis. Use of the PSCA6-2 promoter within the carotenoid pathway engineering experiment increased carotenoid production of target carotenoids by 150% to 197% over production seen from the same constructs run by the promoter PsigA. In addition to engineering of the carotenoid biosynthesis pathway, we also tested the impacts of diel cycle light conditions on carotenoid production and accumulation. When exposed to 12 hour light/dark conditions, the mutant crtR::cruB::ΔcrtO-PSCA6-2::crtW generates carotenoids at rates of 43 ± 14.8 % of that of the same culture grown in constant light conditions. We hypothesized that this lag was caused by the endogenous cellular control of the carotenoid pathway initiated by the metabolic burden placed on the cell. We also hypothesize that this metabolic burden was caused by the engineered constitutive expression of the astaxanthin producing genes during dark conditions. To address potential concerns of constitutive expression of pathway genes during stress conditions like the dark conditions highlighted in the astaxanthin work, our lab constructed a chemically inducible construct for use in Synechocystis that is based on the tac repressor. Upon chemical induction with IPTG, this same mutant strain was capable of exhibiting an average 24X increase in GFP expression over that of the repressed state. In addition to this work, we studied several light induced promoters to better understand their ability to control gene expression during various light conditions in neutral locations within the Synechocystis genome. We identified that the PpsbAII promoter functions very differently in light and dark conditions when it is moved from its native location within the genome. As many researchers utilize this promoter to control gene expression, this information may be critical to fully understanding gene expression of pathways leveraging this promoter construct. Three additional promoter constructs, the PpsbAIII. PgroEL2, and PsigD promoters were also tested for differential expression in light and dark conditions within the neutral region slr0168. Additionally, nucleotide mutations were made to regions within the PpsbAII promoter, to better understand this promoter’s sensitivity to varying light intensities.
Open Access
Nutrient management control regulation and preparedness of a northern Colorado wastewater treatment plant
(Colorado State University. Libraries, 2013) Venkatapathi, Keerthivasan, author; Omur-Ozbek, Pinar, advisor; Carlson, Kenneth, committee member; Reardon, Kenneth, committee member
Excessive nutrients in wastewater treatment plant (WWTP) effluents instigate eutrophication of receiving water bodies. Colorado Department of Public Health and Environment (CDPHE) adopted nutrient management control regulation, also known as regulation 85, to moderate the nutrients released by point sources such as the WWTP effluents. City of Loveland WWTP was selected as the study plant to determine a new treatment process configuration to meet the new limits of total phosphorus < 1 mg/L and total inorganic nitrogen < 15 mg/L in the effluent. BioWin, a windows based modeling software, was used to model and simulate the City of Loveland WWTP. Existing activated sludge step feed process configuration was modeled along with proposed anaerobic, anoxic, oxic (A2O) process for design influent flow of 10 MGD and 5-stage Bardenpho process for future flow of 12 MGD along with A2O process. Existing configuration was modeled to establish the accuracy of BioWin. 5 stage Bardenpho process modeling indicates that higher design HRT of 2 days for anaerobic, 4 days for anoxic, 6 days for aerobic, 4 days for secondary anoxic and 1 day for reaeration has better treatment removal efficiency for nutrients with methanol dosage of 250 gal/d and 1Q internal recycle. Model simulations for A2O process reveals that aerobic reactor to anaerobic reactor volume ratios from 3 to 4 and aerobic reactor to anoxic reactor volume ratio of 2.2 along with internal recycle of 1Q has the better nutrient removal efficiency for design flow of 10 MGD. For 12 MGD influent flow, volume of reactors was increased by 20% to compensate for 20% increase in the flow. Previously mentioned reactor volume ratios are feasible for 12 MGD influent flow with volume ratios of 3 and 4 for aerobic to anaerobic reactors and volume ratios of 1.8 and 2 for aerobic to anoxic reactors. Modeling results indicates that increasing the reactor volume ratio for increased flow can result in better treatment for removal of nutrients with a conservative volume, reducing the operational and maintenance cost.
Open Access
Rewiring anaerobic digestion: production of biofuel intermediates and high-value chemicals from cellulosic wastes
(Colorado State University. Libraries, 2019) Reyes, Jorge L. Rico, author; De Long, Susan, advisor; Sharvelle, Sybil, committee member; Reardon, Kenneth, committee member; Engle, Terry, committee member
Anaerobic Digestion (AD) is a mature biotechnology for the valorization of organic residues, and AD is one of the most popular technologies for organic carbon recovery and waste stabilization. Research and applications for this process have been focused on the production of methane-containing biogas. However, the recent drop in natural gas prices has affected the economic value and market for this biofuel. Existing AD applications for the management of organic wastes (municipal and agricultural) are not economically attractive. Furthermore, it is unclear if methane biogas is the most economically advantageous product. Promising opportunities for AD have emerged in the production of chemical intermediates, such as short-chain fatty acids (SCFA). The market for these chemicals is growing, and more sustainable practices could replace their current petrochemical-based production. AD for the production of SCFA is an alternative approach with attractive market and economic opportunities. This approach is known as the carboxylate platform and relies on the beneficial features of using undefined mixed microbial cultures (also known as microbiomes) for fermentation of heterogeneous organic residues. One of the main identified technological barriers to the carboxylate platform is the inability to control the product spectrum and achieve high yields. AD is a complex biological system, and advances in the fundamental understanding of the microbial ecology associated with SCFA production in these systems are still needed. The identification of specific taxonomic groups involved in the synthesis of certain products could provide insights for novel microbial shaping methods (e.g., bioaugmentation) to improve SCFA selectivity and production yield. This study investigated the relationships between the production of SCFA and the microbial composition from three inoculum sources (anaerobic digester sludge, beef cattle rumen, and bison rumen), with cellulose as a carbon source. Results from the present work found associations between specific taxonomic groups within each of the microbial communities, and the production of particular SCFA. Clostridium lentocellum DSM 5427 and the genus Bacteroides were selectively enriched, and these microbial taxa dominated in anaerobic sludge-inoculated cellulose-fed reactors; these taxa were strongly correlated with acetic acid, caproic acid, and enanthic acid production. On the other hand, propionic acid production was strongly related to the abundance of Prevotella ruminicola, Fibrobacter succinogenes, and members of the family Rikenellaceae. Further investigations at the molecular level (metagenome, metatranscriptome, and proteome) are suggested to expand current knowledge and better understand the microbiological factors that dictate the fermentation of cellulosic material within the context of the carboxylate platform. By expanding this understanding, microbiome shaping methods could be designed and evaluated to optimize and scale-up alternative bioprocessing approaches.
Open Access
Single cancer cell detection with optofluidic intracavity spectroscopy
(Colorado State University. Libraries, 2012) Wang, Weina, author; Lear, Kevin, advisor; Chandrasekar, V., committee member; Krapf, Diego, committee member; Reardon, Kenneth, committee member
The detection of cancer cells is the basis for cancer diagnostics, cancer screening and cancer treatment monitoring. Non-destructive and non-chemical optical methods may help reduce the complexity and cost of related test, making them more available to the public. The label-free technique of optofluidic intracavity spectroscopy (OFIS) uses light transmitted through a cellular body in a microfluidic optical resonator to distinguish different types of cells by their spectral signatures. The OFIS chips are fabricated in the CSU semiconductor clean room and the fabrication process was reported by a previous Ph.D student, Hua Shao. She also did some initial exploration on combining dielectrophoresis (DEP) with the OFIS technique. Since then, some revisions to the fabrication technique have been made to improve the alignment, bonding and sealing of this microfluidic chip. In addition, new DEP electrode designs have been designed and fabricated to further improve the trapping performance of the traps and facilitate automated cell trapping and analysis. Viability tests were carried out to investigate the effect of heating (induced by DEP electrodes) on cells in chips built with borosilicate and sapphire substrates. These experiments used specially designed DEP electrodes that help more accurately control the DEP exposure time and strength. The survival rate of cells out of DEP enabled OFIS system is greatly affected by the substrate type and DEP exposure dose. The OFIS technique has differentiated red and white human blood cells, as well as canine lymphoma and lymphocytes based on their distinctive transmission spectra. Using OFIS chips fabricated with the modified process, OFIS spectra of settled cells from canine hemangiosarcoma (HSA) cell lines and monocytes in peripheral blood mononuclear cells (PBMCs) were collected and analyzed. To quantify the strength of transverse modes in their spectra, a single characteristic parameter was determined for each cell by forming a linear combination of the mean and standard deviation of the transmission spectra over one free spectral range excluding the residual longitudinal peaks of the bare Fabry-Pérot (F-P) cavities filled with cell suspending medium only. The difference in the characteristic parameters of HSA and monocyte samples was highly statistically significant with a p-value as low as 10-6. A receiver operating characteristic (ROC) curve constructed from t-distributions fit to the HSA and monocytes spectra indicates that the cell classification based on their characteristic parameters can achieve 95% sensitivity and 98% specificity simultaneously. Furthermore, some features observed in the spectra of HSA cells motivated a new optical model of the cell loaded F-P cavity. The OFIS spectra of individual cells from canine HSA and canine lymphoma cancer cell lines exhibit relatively uniformly spaced multiple transverse modes repeated in each free spectral range of a microfluidic F-P cavity while similar spectra of healthy canine monocytes and lymphocytes only have up to 2 or no transverse mode peaks. Modeling of the cells as thin lenses allows paraxial Gaussian beam resonator analysis that produces spectral features that quantitatively match the frequencies of transverse modes and qualitatively agree with the trends in maximum transmission of the modes when aperture losses are included. The extracted experimental focal lengths are significantly larger for cancerous cells than for noncancerous cells and can be used as a potential cell malignancy indicator. Furthermore, a thick lens model was developed, allowing manipulation of more parameters related to cell morphology and its location in the cavity. This model was used to interpret experimental results acquired from settled and suspended cells.
Open Access
Sustainability implications of carbon delivery in microalgae cultivation for the production of biofuel
(Colorado State University. Libraries, 2018) Somers, Michael D., author; Quinn, Jason, advisor; Marchese, Anthony, committee member; Reardon, Kenneth, committee member
Supplementation of carbon is critical for high productivity cultivation of most microalgae. Moreover, using microalgae for atmospheric CO2 mitigation to combat climate change is promising, as waste sources and atmospheric CO2 can be utilized to produce useful products. The challenge is developing technologies, processes, and strategies that utilize carbon effectively such that the overall system is sustainable. Through engineering systems modeling combined with techno-economic and life-cycle assessments, this study examined the implications of various delivery methods of carbon to a production-scale algal biorefinery. Five primary carbon sources were considered: atmospheric CO2; CO2 from direct chemical or power plant waste emissions; CO2 that has been concentrated from waste sources and compressed; inorganic carbon in the form of sodium bicarbonate salt; and organic carbon in the form of cellulosic sugars derived from corn stover. Each source was evaluated assuming co-location as well as pipeline transportation up to 100 km. The sensitivity of results to carbon utilization efficiency was also considered. Sustainability results indicate that economics are more prohibitive than energy and emissions. Of the scenarios evaluated, only two met both the economic and environmental criteria of contributing less than $0.50 GGE−1 and 20 gCO2-eq MJ−1 to the overall system, respectively: uncompressed, pure sources of gaseous CO2 with pipeline transportation of 40 km or less; and compressed, supercritical CO2 from pure sources for pipeline transportation up to 100 km. The scalability of algal biofuels based on these results shows carbon to be the limiting nutrient in an algal biorefinery with a total US production capability of 360 million gallons of fuel per year.
Open Access
Sweet sorghum (Sorghum bicolor) biomass, generated from biofuel production, as a reservoir of bioactive compounds for human health
(Colorado State University. Libraries, 2014) Massey, Aaron R., author; Vanamala, Jairam, advisor; Reddivari, Lavanya, advisor; Reardon, Kenneth, committee member
To view the abstract, please see the full text of the document.
Open Access
Synthesis, properties, and suitability of various oxymethylene ethers for compression ignition fuels
(Colorado State University. Libraries, 2023) Lucas, Stephen P., author; Windom, Bret, advisor; Foust, Thomas, committee member; Reardon, Kenneth, committee member; Marchese, Anthony J., committee member
Compression ignition (CI) engines are currently the most common prime mover for medium and heavy duty vehicles; these engines contribute roughly a quarter of US greenhouse gas emissions from transportation, and even higher percentages of particulate and nitrogen oxide emissions. As a result, there have been significant efforts made to reduce these emissions, particularly through selection of low-emissions alternative fuels. Oxymethylene ethers (OMEs) are a class of molecule, typically structured R-O-(CH2O)n-R', which have been considered as a possible blendstock in CI fuels for the goal of soot reduction. Generally, past work has focused on methyl-terminated OMEs, CH3-O-(CH2O)n-CH3, which by virtue of containing no C--C bonds, produce negligible soot. These molecules show significant reductions in soot emission from engines when blended in moderate to high ratios with traditional diesels, however, they have been shown to have inferior physical properties and poor compatibility with some legacy systems. Recent theoretical work has shown that OMEs with non-methyl alkyl groups may have superior performance, albeit at the cost of increased soot formation. In this work, a variety of OMEs with terminating alkyl groups from methyl to butyl are considered for their suitability as CI fuels. The synthesis of these extended OMEs is studied, including formation of n=1 OMEs from common chemical sources, and extension of the chain length to heavier molecules, via reactions over acidic ion exchange resins. Following the synthesis, the properties of these OMEs are studied with respect to their engine applicability. It is found that heavier (propyl- and butyl-terminated) OMEs have superior properties for diesel compatibility, particularly in reactivity, volatility, and water solubility. Extended-alkyl OMEs are found to have higher soot production than methyl-terminated OMEs, but remain superior to diesel soot production on a per-unit-energy basis. A sample of a butyl-terminated OME mixture, n=2-4, is selected as the ideal OME blend for close compatibility with legacy diesel systems. This mixture is blended with certified diesel and tested for ASTM D975 compatibility, passing all required tests but lubricity; decreased heat of combustion is observed but not governed by the diesel standard. Fundamental combustion tests of various mid-weight OMEs are performed in a rapid compression machine, where it is shown that low-temperature chemistry causes a region of decreased dependence of ignition delay on temperature, consistent with methyl-terminated OME behavior. An isopropyl-terminated OME is observed to have low reactivity compared to other OMEs; this fuel is investigated via further rapid compression machine testing and CFR engine testing. It is found that this OME has strong negative-temperature-coefficient ignition behavior - a first for OMEs - and has reactivity lower than other OMEs, but insufficient for direct spark ignition engine testing.
Open Access
The effect of fuel additives in a natural gas and gasoline engine
(Colorado State University. Libraries, 2016) Falloon, Thomas, author; Marchese, Anthony, advisor; Olsen, Daniel, advisor; Reardon, Kenneth, committee member
Fuel additives are used worldwide for a variety of applications including increasing fuel efficiency, decreasing emissions, decreasing knock propensity and/or modifying storage/handling properties. Because of the high percentage of global fossil fuel consumption attributed to internal combustion engines, fuel additives that increase the efficiency of fossil fuel powered internal combustion engines can greatly impact global fossil fuel consumption and greenhouse gas emissions. In this study, the effect of various fuel additives on spark ignited natural gas and gasoline internal combustion engines was examined. The natural gas work focused primarily on using fuel additives to extend the lean limit, while the gasoline additives work focused on lean limit extension, decreased knock propensity and increased power. Experiments were performed in using a constant speed, single cylinder, variable compression ratio Cooperative Fuel Research (CFR) engine, which has the capability to operate with both gaseous and liquid fuels. The gaseous fuel system used compressed air to simulate a turbocharged engine, while the liquid fuel system used a naturally aspirated carburetor. In-cylinder pressure data were acquired using a high-speed piezoelectric pressure transducer, which is used to calculate indicated power, peak pressure and to quantify engine knock. In this study, four natural gas and three gasoline additives were considered. For the natural gas fuel additives, the primary hypothesis for the fuel additives was that the lean limit would be decreased with the addition of the additives. By holding the power of the engine constant and decreasing the equivalence ratio, this hypothesis was tested and it was concluded that the additives had a negative impact on the lean limit. For the gasoline additives, the hypothesis was that the additives would either increase engine power, decrease the knock propensity (i.e. increase the octane number), or decrease the lean limit. It was found that one of the additives increased engine efficiency slightly and decreased the knock propensity, while the other two gasoline additives had negative impacts on both metrics. One of the gasoline additives appeared to slightly extend the lean limit, but further testing will be required to confirm this result.