Browsing by Author "Reardon, Kenneth F., advisor"
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Item Open Access Bioconversion of lipid-extracted algal biomass into ethanol(Colorado State University. Libraries, 2016) Mirsiaghi, Mona, author; Reardon, Kenneth F., advisor; Peebles, Christie, committee member; Peers, Graham, committee member; Smith, Gordon, committee memberEnergy security, high atmospheric greenhouse gas levels, and issues associated with fossil fuel extraction are among the incentives for developing alternative and renewable energy resources. Biofuels, produced from a wide range of feedstocks, have the potential to reduce greenhouse gas emissions. In particular, the use of microalgae as a feedstock has received a high level of interest in recent years. Microalgal biofuels are promising replacement for fossil fuels and have the potential to displace petroleum-based fuels while decrease greenhouse gas emissions. The primary focus of research and development toward algal biofuels has been on the production of biodiesel or renewable diesel from the lipid fraction, with use of the non-lipid biomass fraction for production of biogas, electricity, animal feed, or fertilizer. Since the non-lipid fraction, consisting of mainly carbohydrates and proteins, comprises approximately half of the algal biomass, our approach is biological conversion of the lipid-extracted algal biomass (LEAB) into fuels. We used LEAB from Nannochloropsis salina, and ethanol was the model product. The first step in conversion of LEAB to ethanol was deconstruction of the cell wall into fermentable substrates by using different acids or enzymes. Sugar release yields and rates were compared for different treatments. One-step sulfuric acid hydrolysis had the highest yield of released sugars, while the one-step hydrochloric acid treatment had the highest sugar release rate. Enzymatic hydrolysis produced acceptable sugar release rates and yields but enzymes designed for algal biomass deconstruction are still needed. Proteins were deconstructed using a commercially available protease. The hydrolysate, containing the released sugars, peptides, and amino acids, was used as a fermentation medium with no added nutrients. Three ethanologenic microorganisms were used for fermentation: two strains of Saccharomyces cerevisiae (JAY270 and ATCC 26603) and Zymomonas mobilis ATCC 10988. Ethanol yields and productivities were compared. Among the studied microorganisms, JAY270 had the highest ethanol yield while Z. mobilis had the lowest yield for most of the studied conditions. A protease treatment improved the biomass and ethanol yields of JAY270 by providing more carbon and nitrogen. To increase ethanol productivity, a continuous fermentation approach was adapted. Continuous stirred tank reactors have increased productivity over batch systems due to lower idle time. The downtime associated with batch fermentation is the time it takes for empting, cleaning, and filling the reactor. Productivity in the continuous fermentation was limited by the growth characteristics of the microorganism since at high flow rates, with washout occurring below a critical residence time. To overcome the washout problem, the use of an immobilized cell reactor was explored. The performance (ethanol productivity) of free and immobilized cells was compared using an enzymatic hydrolysate of LEAB. Higher ethanol productivities were observed for the continuous immobilized cell reactor compared to the stirred tank reactor.Item Open Access Biodegradation of dinitrotoluene by Pseudomonas PR7 in a fluidized bed bioreactor(Colorado State University. Libraries, 1997) Whitty, Katherine Keesling, author; Reardon, Kenneth F., advisor; Murphy, Vincent G., committee member; Linden, James C., committee member2,4-Dinitrotoluene (DNT) has been listed as a priority pollutant by the U. S. EPA. It is a waste product in the production of 2,4,6- trinitrotoluene (TNT) and toluene diisocyanate. Pseudomonas PR7 is able to completely degrade DNT via an oxidative pathway. Batch suspended-cell experiments were performed in order to determine the maximum specific growth rate Pmax/ and the Monod half-saturation constant. Ks. Parameter values of μmax = 0.1 h-1 and Ks = 14 mg/L were obtained by fitting experimental data to the Monod model. Immobilized-cell experiments in a fluidized-bed bioreactor (FBB) were performed in order to determine volumetric DNT degradation rate v for the biodegradation of DNT. A fluidized-bed bioreactor was chosen for study because (1) immobilization of cells onto particles allows for greater cell retention, and (2) fluidization of particles allows for mixing within the reactor. Greater cell retention allows for higher flow rates of liquid through the reactor and adequate mixing can alleviate the problem of low oxygen availability and other accumulation or depletion problems which occur in packed beds. Fluidization of immobilized cells in the FBB was achieved by the upflow of air and liquid. Data from residence time distribution (RTD) analysis of the FBB suggests that it behaves as a stirred tank reactor with small plug-flow regions and dead zones. The fluidized-bed bioreactor performance was compared with that of suspended-cell experiments and packed-bed experiments through direct comparison of DNT loading versus degradation rates. It was found that the fluidized-bed bioreactor performed as well as a previously reported system consisting of a packed-bed column in series with a stirred-tank reactor in one experiment using diatomaceous earth particles as the immobilization medium. The FBB did not perform as well as the packed-bed system in subsequent experiments using polycarbonate particles.Item Open Access Crop protection in industrial algae farming: detecting weedy algae and characterizing bacterial communities(Colorado State University. Libraries, 2015) Fulbright, Scott Paul, author; Reardon, Kenneth F., advisor; Reddy, Anireddy, committee member; Laybourn, Paul, committee member; Wallenstein, Matthew, committee member; Tisserat, Ned, committee memberMicroalgae are a promising source of feedstock for biofuel and bioproducts. Algae have higher rates of biomass production than terrestrial crops, and therefore can use less land for producing equivalent energy compared to other biofuels. Elite algae strains are chosen based on traits such as fast and robust growth, and rapid production of desired biochemical products, including fatty acids and other high-energy compounds. Monocultures of elite strains are grown in large algae production systems. A major challenge algae growers face is consistently growing robust cultures of elite algae. This is due to unwanted organisms invading cultures such as weedy algae that contain less desirable biochemical products, and bacteria that can detract from algae growth, thereby reducing overall system productivity. Historically, algae have not been grown at scales required for biofuels and bioproducts, and thus there is a lack of fundamental pest management knowledge and developed tools. In this work, we developed three polymerase chain reaction (PCR)-based tools for detecting and quantifying weedy and elite algae. We developed a simple and inexpensive CAPS (cleaved amplified polymorphic sequence) assay that can determine the presence of dominant algae species in cultures. Also, we developed and validated qPCR primers were able to detect one weedy algae cell in 108 cells in a culture. Compared to flow cytometry, the qPCR primers were 104 times more sensitive for detecting weedy algae. We validated tools by monitoring industrial algae systems, and exhibited their utility for assisting in culture management decisions. Bacteria are also prevalent in industrial algae cultures yet little is understood about their dynamics or role in the ecosystem of elite algae cultures. We sampled small, medium and large cultures from an industrial algae system growing elite algae Nannochloropsis salina, and sequenced the 16S rDNA gene and used QIIME bioinformatics program to analyze data. In this study, we characterized bacterial communities diversity, richness, and composition in industrial algae bioreactors during the scale-up process, through time and during various algae growth rates. We demonstrate that bacterial diversity richness increases as the size of the algae production system increases in the scale-up process. Therefore, larger cultures are comprised of more complex communities than smaller cultures, thus increasing the probability of detrimental algae-bacteria interactions. We identified a single core bacterium Saprospiraceae that was present in 100% of samples, and was on average the most abundant bacterium in all systems. Further, we identified a Deltaproteobacterium that was detected at abnormally high relative abundances in poorly growing algae cultures. Identifying pest bacteria that can detract from elite algae growth is an important step in developing crop protection strategies. We isolated bacteria from a poorly performing algae system and determined their influence on algae growth. We identified a single isolate, S7 as a growth inhibiting bacteria that was capable of completely inhibiting Nannochloropsis gaditana and N. salina growth. The bacterium was characterized as Bacillus pumilus. Additionally, we identified nutrients and cell concentrations required for inhibition of N. gaditana and N. salina. B. pumilus inhibition effect is species-specific as it did not inhibit weedy algae, Chlorella vulgaris and Tetraselmis striata. Due to this, B. pumilus is capable of manipulating algae population composition and reducing productivity. Contaminating organisms such as bacteria will often be prevalent in algae systems and understanding their influence on culture productivity is essential for successful large-scale cultivation of algae. In summary, we 1) developed molecular tools to monitor weedy algae that can be used by growers, 2) characterized bacterial communities in industrial algae system cultures, and 3) identified a novel pest for elite algae, N. gaditana and N. salina.Item Open Access Development and application of functional gene profiling and quantification of microbial communities remediating mine drainage(Colorado State University. Libraries, 2009) Pereyra, Luciana Paula, author; Pruden, Amy, advisor; Reardon, Kenneth F., advisorMine drainage (MD) is the product of the oxidation of sulfide minerals. It is characterized by elevated concentrations of heavy metals and sulfate and acidic to near-neutral pH. Sulfate-reducing permeable reactive zones (SR-PRZs) represent a common passive treatment approach for MD. Although SR-PRZs are microbially catalyzed, little is known about their microbiology and ecology. In this research, several aspects of the SR-PRZ microbial community were explored at laboratory and pilot scales with established as well as newly developed biomolecular methods. A study using microcosm column experiments demonstrated that the type of inoculum plays an important role in the bioremediation of MD. The effect of the type of substrate on the microbial community was also investigated in pilot-scale SR-PRZs treating the MD. Lignocellulose-based SR-PRZs contained a more diverse microbial community and higher bacterial density than ethanol-fed SR-PRZs, as determined by 16S rRNA gene cloning and quantitative polymerase chain reaction (Q-PCR). A new biomolecular approach was developed to target genetic markers of the functions of interest (functional genes): cellulose degradation, fermentation, sulfate reduction, and methanogenesis. This approach provided a more efficient and direct means of studying microbial functions. The functional gene-based approach was adapted to denaturing gradient gel electrophoresis and Q-PCR and applied to study the microbial communities in laboratory columns simulating SR-PRZs during the initial and pseudo-steady-state operation. Although the microbial communities in the different treatments were different during pseudo-steady-state operation, performance of the columns was comparable in terms of sulfate and metal removal and pH neutralization. This suggests that various microbial compositions can lead to successful MD remediation. The studies presented in this dissertation provide significant insight in the microbial communities involved in MD remediation at laboratory and pilot scale. In addition, a variety of biomolecular methods are presented that can be applied to explore different aspects of the microbial community not only in SR-PRZs and but also in other systems with complex microbial communities. Integration of biomolecular and performance data will provide a more complete understanding of SR-PRZ function that could be used to improve SR-PRZ performance and reliability.Item Open Access Development and application of microbial community profiling techniques for mine drainage bioremediation(Colorado State University. Libraries, 2008) Hiibel, Sage Royal, author; Reardon, Kenneth F., advisor; Pruden, Amy, advisorAcid mine drainage (AMD), characterized by elevated levels of sulfate, acidity, and metals, is produced by the oxidation of mining-exposed minerals and is a major environmental issue. Sulfate-reducing bioreactors (SRBRs) are an attractive AMD treatment option. SRBRs contain an organic material, usually wood chips or compost, which provides a slow-release carbon substrate to support a complex anaerobic microbial community. A relationship between the microbial inoculum and bioremediation performance was established in laboratory experiments. The use of 16S rDNA-based profiling techniques established a correlation between SRBRs that performed well and the presence of three key functional groups: cellulose degraders, fermenters, and sulfate-reducing bacteria (SRB). Subsequent analyses of pilot- and field-scale SRBRs targeted the 16S gene and apsA functional gene, which is found in all SRB. Although multivariate statistical analyses of the 16S sequences of the communities did not reveal obvious differences, the apsA sequences of each SRBR were significantly different. The apsA sequences also revealed that Thiobacillus spp., which are capable of sulfur oxidation, were prevalent at the poorly performing SRBR. A novel, high throughput, biomolecular method called active community profiling (ACP) was developed and validated using model systems. ACP identifies the active members of mixed communities through the ratio of rRNA to rDNA, which is proportional to growth rate. When coupled with physiochemical analysis, ACP offers a powerful new tool to help understand microbial community dynamics. The effects of bioaugmentation and biostimulation on the community structure of AMD treatment systems were studied. Although all columns remediated AMD to a similar level, ACP analysis revealed that the active members of their communities were distinctive. It was determined that biostimulation or bioaugmentation at the top of the microbial carbon chain increased the active community diversity. This dissertation emphasizes the role of the microbial community associated with AMD remediation. Characterization of these communities with biomolecular tools at several scales has significantly advanced the understanding of the community's structure, function, and activity. The research approaches and methodologies developed have wide application, and provide unique and valuable contributions to the scientific knowledge of AMD treatment specifically, and to microbial ecology and bioremediation in general.Item Open Access Environmental proteome profiling applied to the study of polybacterial metal resistance and adaptation(Colorado State University. Libraries, 2008) Lacerda, Carla M. R., author; Reardon, Kenneth F., advisorEnvironmental biotechnology can be defined as the use of biotechnology to solve environmental engineering problems, frequently involving bacterial communities and unsequenced species. Here we define environmental proteomics as the proteomic profiling of microorganisms of environmental relevance, targeting the improvement of environmental bioprocesses. This study demonstrates our ability to obtain proteomic data for communities of microorganisms and for environmental isolates, providing unique insights into the physiology and ecology of these systems. A combination of qualitative and quantitative proteomics methods (two-dimensional electrophoresis and/or chromatography followed by tandem mass spectrometry) was used to investigate the proteome of a sequenced mixed culture, an unsequenced mixed culture, and a bacterial isolate from the original unsequenced mixed culture. In the first case study, two soil organisms were grown in co-culture in an attempt to observe proteins induced as a response to the presence of another organism. Many proteome changes were detected and quantified, with proteins involved in protein and DNA metabolism being the most largely modulated. In the second case study, an unsequenced mixed culture was exposed to cadmium and had its dynamic response analyzed. While the community responded significantly to all shock durations, the greatest amount of change was observed in the first fifteen minutes of shock. The main groups of differentially expressed proteins identified were transport proteins, showing that the main method for cadmium tolerance was active efflux. In the study of the adaptation of a pure culture, the most cadmium-tolerant organism in the original unsequenced community was isolated and cultivated in different concentrations of cadmium. In the last case study of metal resistance, the proteomes of this isolate were compared as it responded to short-term exposures to chromium, iron and cadmium. Metals induced proteome responses in both short- and long-term exposures, meaning that the mechanisms for adaptation and resistance are different. This project demonstrates the potential of environmental proteomics and its intricacies as different proteomic workflows are employed. This is also one of the first evaluations of metaproteomic changes due to the metal response of mixed bacterial cultures, revealing the large potential of environmental proteomics to uncover unique insights into systems-level bacterial functions.Item Open Access Fiber optic enzymatic biosensors and biosensor arrays for measurement of chlorinated ethenes(Colorado State University. Libraries, 2011) Zhong, Zhong, author; Reardon, Kenneth F., advisor; Lear, Kevin, committee member; Dandy, David S., committee member; Henry, Charles S., committee memberChlorinated ethenes such as trichloroethylene (TCE), tetrachloroethylene (PCE), three isomers of dichloroethylene (DCEs) and vinyl chloride (VC) are used as solvents and cleaners in a variety of industrial and commercial areas. Chlorinated ethenes have become one of the most common environmental pollutants in groundwater contamination sites due to their widespread usage, moderate solubility compared with other organic pollutants and recalcitrance to natural attenuation. Fiber optic enzymatic biosensor was developed in this study as a continuous, real time and in situ measurement principle. TOM biosensor, first reported enzymatic biosensor, was initiated with toluene measurement in aqueous solution as proof-of-concept experiments. The subsequent success of TOM and TOM-Green in TCE analysis showed great potential of biosensor measurement for chlorinated ethenes, despite the ubiquitous problem for monooxygenase-based biosensor with NADH consumption overtime and after usage. In addition, epoxide toxicity also increased the difficulty of biosensor application for measurement of chlorinated ethenes, although several TOM-Green transformants could mitigate the toxicity with rapid epxoide degradation. Plasmid transformation with was introduced to manipulate the construction of new TOM and TOM-Green transformants with capability of intracellular NADH regeneration. FDH regeneration system was studied for both TOM and TOM-Green cells, while TOM+FDH showed great activity retention and regeneration ability and TOM-Green+FDH was able to retain activity over prolonged storage but failed on regeneration after repeated usage due to the toxicity of TCE epoxide. Biosensor array was built with pH-based biosensor to measure a group of haloalkanes. The design concept of biosensor array and detection instrumentation was successful. Linear approach in array data analysis was simple and fast but lacked of accuracy, while nonlinear approach increased the complexity of data analysis to a new level with precision in sacrifice of efficiency. Multivariable chemometric approach was also introduced in array data analysis, providing a high-throughput alternative and a means of quantitatively assessing matrix effects. This project demonstrates the potential of fiber optic enzymatic biosensor and biosensor array as measurements for different analyte are described. This is also one of the first comprehensive studies in oxygen-based biosensor and its application and great potential in food, clinical, and environmental monitoring, industrial process control and other related areas.Item Open Access Immunoproteomic identification of bovine pericardium xenoantigens(Colorado State University. Libraries, 2008) Griffiths, Leigh G., author; Orton, E. Christopher, advisor; Reardon, Kenneth F., advisorBovine pericardium (BP) is an important biomaterial used in the production of gluteraldehyde-fixed heart valves and tissue engineering applications. The ability to perform proteomic analysis on BP is potentially useful for several reasons including investigation of immune rejection after implantation. The importance of humoral and cell mediated rejection responses towards such xenogeneic tissues are becoming increasingly apparent. I have applied a novel immunoproteomic approach to survey the antigenic determinants of BP. Proteomic analysis of fibrous tissues like BP is challenging due to their relative low cellularity and abundance of extracellular matrix. A variety of methods for tissue homogenization, protein extraction, and fractionation were investigated with the aim of producing high quality 2-DE gels for both water- and lipid-soluble BP proteins. MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular fractionation of resolved proteins. Sixteen unique predominantly cytoplasmic bovine proteins were identified from the water-soluble gels. Twenty-two unique predominantly membrane bovine proteins were identified from the lipid-soluble gels. These results demonstrate that the final 2-DE protocol produced high quality proteomic data from BP for both cytoplasmic and membrane proteins. Duplicate 2-DE gels were used to generate western blots from both water- and lipid-soluble gels. Western blots were probed with pre- and post-exposure anti-BP rabbit serum, with detection of immune complexes limited to the IgG subtype. Western blots were compared to duplicate 2-DE gels and spots matched using Delta 2D image analysis software. Protein identifications of matched spots were performed using either MALDI-TOF/TOF MS or ESI MS/MS. This approach identified 31 putative antigens, capable of stimulating an IgG humoral rejection response. To the best of my knowledge, this study was the first to apply an immunoproteomic approach for identification of antigenic targets in xenotransplanted tissues. The results provide important information for understanding and possibly mitigating the immune response to fixed and unfixed BP xenografts.Item Open Access Initial development of a multistage cancer model based on Syrian hamster embryo (SHE) cell transformation studies(Colorado State University. Libraries, 1999) Liao, Kai-Hsin, author; Yang, Raymond Shih-hsien, 1940-, advisor; Reardon, Kenneth F., advisor; Murphy, V. A., committee memberTo better incorporate biologic information into quantitative cancer modeling, the two-stage MVK (Moolgavkar-Venzon-Knudson) model has been modified for use with SHE cell neoplastic progression. Conceptually, five phenotypic stages are included in this model: normal cells can either become senescent or mutate into immortal cells followed by anchorage-independent growth and tumorigenic stages. Cells in each stage have distinct division, death and mutation rates, and mutation is assumed to occur during cell division. Model development and related experiments were focused on studying the abilities of lead, arsenic, chromium, and a mixture of these three metals to induce progression of SHE cells from one phenotype to the next. Cell division and death rates were assessed using flow cytometric analysis for inclusion in the model. Cell division rates were measured using bromodeoxyuridine (BrdU) incorporation with propidium iodide staining, which allows for the calculation of potential doubling time, a measure of cell cycle time that takes growth fraction, but not cell loss, into account. Potential doubling times of normal SHE cells ranged from 12 to 59 hours, depending on the degree of confluence of cell cultures. Cell death was measured by a flow cytometry method based on propidium iodide staining specifically related to membrane damage. The mean cell death rate is approximately equal to 1 % of the average value of division rates. The individual metals and their mixture did not induce immortalization or further mutations of SHE cells in our laboratory following a 2-day exposure. However, the growth of SHE cells was inhibited by 5.4 μM of arsenic, with cells becoming senescent after only 16 population doublings; whereas, normal cells and cells exposed to lower arsenic concentrations lasted for at least 30 population doublings. The model developed in our laboratory successfully predicted the growth of normal cells. The cell senescence rates under the impact of arsenic exposure were also calculated. Mechanisms responsible for induction of cellular senescence in SHE cells exposed to arsenic may be involved in the apparent inability of arsenic to induce neoplasia in experimental animals.Item Open Access Investigation of the inhibitory effect of Bacillus pumilus on Nannochloropsis salina(Colorado State University. Libraries, 2017) Ayshoa Al Gabara, Mirna Dheyaa, author; Reardon, Kenneth F., advisor; Argueso, Cris, committee member; Peebles, Christie, committee memberMicroalgae have the potential to be a source of a wide range of industrial materials. To provide the biomass for these products, algae are grown in large volumes. Previous research has shown that there are other microbial species living in algal cultivation systems at these scales, but little is known about the interactions among them. Some of the bacteria in algae cultivations have been identified. Some species can inhibit algal growth, while others are growth promoting. In this research, we focused on one algal species, Nannochloropsis salina, and a bacterial species, Bacillus pumilus. In previous research in our laboratory, B. pumilus culture filtrate had inhibitory effects towards N. salina. We are using these species as a model system to understand a mechanism of bacterial inhibition of algae. Specifically, we have investigated the nature of the inhibitory molecule that is produced by B. pumilus and when it is produced. Our results indicate that B. pumilus produces at least one inhibitory molecule that is probably a protein larger than 30 kD. Since the bacteria produce the highest level of the inhibitory molecule in the presence of marine broth medium (MB), we studied the effects II of the components of MB to determine whether one of these induced the production of the inhibitor more than others. B. pumilus was inoculated in artificial sea water medium (ASW) and several components of MB (peptone, yeast extract and glucose). The filtrate of B. pumilus grown in ASW supplemented with peptone or yeast extract had an inhibitory effect on N. salina, but the filtrate of B. pumilus grown in ASW supplemented with glucose had no inhibitory effect towards the algal species. The results showed that the molecule was produced regardless of the presence of the algal species and it was more concentrated at the late stationary phase. Also there was a certain algal phase when N. salina had more resistance to the inhibition of B. pumilus filtrate. The bacterial species showed the ability to grow on the filtrate of N. salina without any other added components. This knowledge about the mechanism by which this bacterial species inhibits an algae species is useful to determine whether other bacteria use the same strategy and to develop an approach to reduce this inhibitory impact.Item Open Access Nitrogen recovery from anaerobic digestate via ammonia stripping and absorbing with a nitrified solution(Colorado State University. Libraries, 2021) Alhelal, Ismail Ibrahim, author; Reardon, Kenneth F., advisor; Sharvelle, Sybil, advisor; Perkins, Tracy, committee member; Carlson, Kenneth, committee memberAnimal wastes cause environmental pollution, including contamination of air and water, when not managed properly. For example, stored livestock manure releases greenhouse gasses, which contribute to air pollution and global warming. Anaerobic digesters have been used for animal waste treatment in order to reduce the environmental impacts of animal wastes. However, current anaerobic digestion systems have serious economical and operational challenges such as high capital cost, low byproduct price, and ammonia toxicity. Therefore, more research is needed to increase the benefits of anaerobic digestion and reduce its challenges. The goal of this project was to improve the cost and performance of anaerobic digesters by enhancing their byproducts, biogas and fertilizer, while reducing one of their serious operational challenges, ammonia toxicity. To achieve these goals, this project investigated an integrated anaerobic digestion nitrogen recovery process that includes anaerobic digestion, nitrogen recovery and nitrification. The nitrogen produced during anaerobic digestion is volatilized in a stripper, captured in an absorber, and converted to nitrogen certified organic fertilizer in the nitrification process. Recovering the ammonia in anaerobic digesters not only produces organic fertilizer but also reduces ammonia toxicity, enhancing biogas production. Experiments and modeling were used to identify appropriate operating conditions for the stripper and absorber units of the proposed process. The objective of the nitrogen recovery system experiments was to find the best operational conditions as well as to evaluate the performance of the nitrification solution as an ammonia absorbent. Stripping and absorption columns were designed to measure the ammoniacal nitrogen recovery. The ammonia stripping and absorption extents were calculated for several operational conditions: stripping and absorption feed pH, stripping temperature and absorbent nitrogen concentration. The experimental results showed that a feed pH of 10 was optimal for ammonia stripping in the pH range 8.5–10.5, providing an ammonia stripping extent of 77%, while the optimal stripping temperature was 50 °C since it provides the highest extent of ammonia stripping in the tested range of 35–65 °C. An Aspen Plus simulation model was also developed for the ammonia stripping process to calculate the effects of the number of equilibrium stages, feed pH, and the amount of CO2 in the stripping gas. The model showed that the use of three equilibrium stages, a feed pH of 10, and having no CO2 in the stripping gas provides the most feasible operational conditions considering the stripping performance and economics. Moreover, the data suggested that the stripping units will require pH control for effective ammonia recovery since the pH of the stripper decreases with the ammonia removal. For the ammonia absorption unit, the experimental data showed that ammonia absorption was not greatly impacted by the feed pH nor by the concertation of nitrogen in the liquid feed. With a low concentration of nitrogen in the liquid feed (2 g/L NH4NO3 as N), the extents of ammonia absorption for feed pH values of 7 and 2 were 82% and 92, respectively. However, the extents of ammonia absorption using a high concentration nitrogen liquid feed (7 g/L NH4NO3) for feed pH values of 7 and 2 decreased to 70% and 85%, respectively. However, a Two-Factor ANOVA test with replication has a p-value >0.05, so there is no statistically significant difference in the ammonia absorption due to the feed pH nor in the concentration of nitrogen in the absorbent. Consequently, it can be concluded that nitrified solution can be used as an ammonia absorbent because it can affectively absorb ammonia over a wide range of its pH and its nitrogen concentration. This project demonstrated that it is possible to recover nitrogen in an integrated anaerobic digestion process and determined recommended operational conditions for the nitrogen recovery system. The novel integrated anaerobic digestion system proposed in this work decreases ammonia toxicity for anaerobic digestion, while increasing potential for revenue from increased biogas yield and recovery of ammonia fertilizer. increasing the biogas yield, producing organic fertilizer and decreasing ammonia toxicity.Item Open Access Nitrogen utilization in heterotrophic Chlamydomonas reinhardtii(Colorado State University. Libraries, 2017) Sweeley, Justin Brye, author; Reardon, Kenneth F., advisor; Peebles, Christie, committee member; Snow, Christopher, committee member; Peers, Graham, committee memberThe aim of this dissertation research is to bring better understanding to the process of nitrogen adaptation in heterotrophic Chlamydomonas reinhardtii. Microalgae are a diverse group of aquatic photosynthetic organisms that account for almost 50% of the photosynthetic productivity on Earth. There is immense interest in using the unique ability of microalgae to convert sunlight to triacylglycerides (TAGs) for industrial purposes. However, to date there has been little success in implementing these systems at scale and price parity with non-biological methods. Microalgae can modify their metabolism to adapt to the surrounding environment. Under certain circumstances, including nutrient stress, microalgae divert carbon flow away from biomass production and into TAG accumulation. The most common nutrient stress used to trigger TAG accumulation is nitrogen stress, most often induced by transferring a cell from a nitrogen replete medium to a deficient one. The goals of this research were to understand this process, develop methods to manipulate the stress response, and ultimately, to find a way to decouple lipid production from nutrient depletion entirely. Chapter 1 introduces the concepts and research referenced throughout the dissertation including: a background of the C. reinhardtii species, the cultivation techniques that have been applied to cultivation, the physiology behind nitrogen stress, the mechanism that algae use to incorporate nitrogen into the cell, and finally an introduction to the global nitrogen regulator, PII. Chapters 2 through 4 present research into the nitrogen stress pathway and its modification. Chapter 2 discusses a simple method of cultivation used to bring about new insights into the nitrogen stress response, as well as a proposed technique for increasing cellular lipid production. Through differential nitrogen feeding, significantly different effects on cell growth were observed, demonstrating that the response to nitrogen availability is a continuous effect as opposed to an all or nothing "stress response". Chapter 3 describes experiments in which C. reinhardtii was genetically modified to increase understanding of the nitrogen stress response. A nitrogen regulatory protein, PII, was downregulated via amiRNA. Cultures of a mutant strain with lower levels of PII exhibited slow adaptation to fresh nutrient-replete medium but achieved a higher final cell number, final mass concentration, and total neutral lipid content. Similar results were obtained in cultures shifted to nitrogen-free medium. Chapter 4 employs proteomics to identify differences in the specific protein expression pattern between a functional PII strain and a knock-down mutant. Chapter 5 demonstrates a unique approach to producing an engineered nutrient-limited environment in a continuous stirred tank bioreactor. Chapters 7 and 8 summarize the research findings and offer possible direction for future research. Through this research work, new information was obtained on the effects of PII on the cellular response to nitrogen limitation. By increasing our understanding of this basic mechanism, we have proposed several processing conditions that may be implemented to increase microalgal productivity. Furthermore, the homology between microalgae and terrestrial plants suggests the possibility that the results discussed within could give genetic engineers new targets for creating crops with decreased nitrogen demands and increased nitrogen-stress tolerance traits.Item Open Access Reaction network model for the prediction of mammalian metabolism of benzo[a]pyrene(Colorado State University. Libraries, 2004) Liao, Kai-Hsin, author; Yang, Raymond Shih-hsien, 1940-, advisor; Reardon, Kenneth F., advisor; Dandy, David S., committee member; Andersen, Melvin E., committee memberHumans are exposed to mixtures of environmental pollutants on daily bases. Many of these chemicals undergo biotransformation in our body and often produce toxic metabolites. The biotransformation of mixtures involves complex reaction networks that are difficult to study using conventional experimental techniques. As a first step of developing a predictive tool for the biotransformation of chemical mixtures, a chemical engineering approach, Reaction Network (RN) modeling, was utilized to study the mammalian metabolism of benzo[ a ]pyrene (BaP), a priority environmental carcinogen. A RN pathway model which predicts the theoretically possible reaction network for BaP was first developed based on the existing modeling technology for predicting the reaction networks in petroleum refinery processes, mechanistic organic chemistry, as well as the commonly observed biochemical reactions for mammalian metabolism of BaP. The resulting RN pathway model for BaP predicts that 246 reactions can occur, resulting in unique 150 products in the presence of mammalian cytochrome P450 and epoxide hydrolase. Some of these predicted products might not be experimentally detected due to the slow reactions for their formation or the production of reactive species. A RN kinetics model which reflects the experimentally measurable metabolic pathways was then established to determine the reaction rates of BaP metabolism. To obtain proper separation of eleven BaP metabolites with high detection sensitivities, high-performance liquid chromatography methods were developed and validated. The RN kinetics model was calibrated and validated using experimental data of BaP metabolism catalyzed by recombinant human enzymes. The biotransformation of BaP and the production of nine BaP metabolites were accurately described by the RN kinetics model. Finally, the RN kinetics model of BaP was linked to a physiologically based pharmacokinetic (PBPK) model to describe the distribution and disposition of BaP and its metabolites in rats. The major advantages of applying RN modeling to study toxicology are: (1) their capabilities of handling complex metabolic systems; (2) their potential for predicting reaction networks of chemicals with limited knowledge on their metabolic pathways; and (3) their abilities to predict the reactive intermediates that are not readily measurable in experiments.Item Open Access Using poppy oil for energy in Afghanistan: a life cycle assessment and diffusion of innovations approach toward sustainable livelihood development(Colorado State University. Libraries, 2014) Mourning-Star, Phoenix, author; Reardon, Kenneth F., advisor; Baron, Jill, advisor; Galvin, Kathleen, committee member; Peek, Lori, committee memberTo view the abstract, please see the full text of the document.