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The fermentation of syngas (a mixture of CO, CO2 and H2) to produce ethanol is of interest as an alternative fuel. Clostridium carboxidivorans, has been found to produce higher than average amounts of ethanol and butanol from CO-rich mixtures. This project sought to determine the effects of the redox level in the solution, partial pressures in the headspace and mass transfer limitations on the products obtained through fermentation of syngas. It was determined that cysteine sulfide has a greater effect on the redox level of the media used to grow bacteria, than does the gas composition. Therefore, changing gas composition during the process will have little effect on the redox. However, addition of cysteine sulfide may vary the redox level. When cells were first inoculated, the redox level dropped and leveled at -200 mV SHE for optimal growth. In addition, cells switch from acetic acid to ethanol production after a drop of 40-70 mV in the redox level. Different sizes of reactors were used, including 1 liter reactors (non-pressurized), 50 mL bottles (20 psig) and 100 mL bottles (20 psig). The 50 mL bottles have more than double the growth rate than the 100 mL bottles (0.57 day-1 compared to 0.20 day-1). Partial pressures were measured in these two sizes to determine the different consumptions and the effect of partial pressure on both growth and production of acetic acid/ethanol. It is clear that re-gassing the bottles every 12 hours to keep the pressure higher in the 100 mL bottles makes a significant difference in the growth, making them very similar to the 50 mL bottles. Both the 50 mL and 100 mL bottle were found to have essentially the same mass transfer rate (0.227 L/hr vs. 0.255 L/hr). However, because of headspace differences, there was more CO available for the 50 mL bottles (on a per liter basis) as compared to the 100 mL bottles. Mass transfer analysis proved useful in pointing out that all three reactors likely experienced mass transfer limitations such that mass transfer effects are critical to address when performing studies involving syngas fermentation.

Ethanol usage as a transportation fuel is rapidly increasing in the United States. Production of ethanol from cellulose feedstocks via gasification followed by syngas fermentation offers an environmentally friendly approach that mitigates many of the adverse effects associated with production from corn. In the syngas fermentation process, the hydrogenase enzyme of the fermentation bacterium, Clostridium P11 for this work, supplies electrons to the metabolic pathway, facilitating ethanol production. In this thesis, an assay for P11 hydrogenase activity was developed. It was determined that 1) less than 4 minutes of sparging with 50 sccm H2 is needed to reduce O2 levels to below 1 ppm in a 3 mL aqueous solution, while less than 1 minute of purging at the same rate is needed to fill an air-filled 3.5 mL cuvette to 99.9999% H2, 2) 12.5 mM DTT included in the reaction mixture at pH 6 helps scavenge O2, 3) H2 diffusion is slow compared to enzymatic reaction rates, 4) CO2 lowers media pH, 5) 0.084 atm CO causes 90% inhibition of P11 hydrogenase, 6) prolonged Triton X-100 exposure diminishes hydrogenase activity, and 7) variations in H2 pressure and electron acceptor identity and concentration affect measured hydrogenase activities. The assay developed for P11 hydrogenase activity was used to perform kinetic studies. The Okura rapid-equilibrium rate law best described this activity. A constant that regulates the effect of H2 pressure on hydrogenase activity, KH2, was determined to be independent of electron acceptor and to have a value of 0.31 atm, implying that H2 must be supplied to the syngas fermentation at ~3 atm to maximize hydrogenase activity. KBV and KMV, constants that regulate the effect of benzyl viologen and methyl viologen on hydrogenase activity, were determined to be 1.7-2.4 mM and 10.6 mM, respectively. Additionally, hydrogenase activity was temporally correlated with ethanol production in batch cultures of P11 and strongly dependent on pH. The intracellular pH of P11 was determined to be approximately 5.5.

A syngas compositional database with focus on trace impurities was established. For this work, ammonia (NH3) and benzene (C6H6) effects on cell growth, enzymatic activities of hydrogenase and alcohol dehydrogenase (ADH), and product formation were studied. NH3, after entering media, will be converted rapidly to NH4+, which will raise the total osmolarity of the media. NH3, as a common nutrient for the cell growth, is not the real culprit for cell growth inhibition. In essence, it is the high osmolarity resulting from the accumulation of NH4+ in the media which disrupts the normal regulation of the cells. It was concluded that at NH4+ concentration above 250 mM, the cell growth was substantially inhibited. However, P11 cells used in this study can likely adapt to an elevated osmolarity (up to 500 mM) although the mechanism is unknown. It was also found that higher osmolarity will eventually lead to higher ethanol per cell density. In conclusion, NH3 needs to be cleaned out of syngas feeding system. The realistic C6H6 concentration in the media coming from a gasifier was simulated in bioreactors and was measured by a GC/MS. The most realistic C6H6 concentration in the media was around 0.41 mM (upper limit 0.83 mM). However, five elevated concentrations of 0.64, 1.18, 1.72, 2.33, and 3.44 mM were doped into the media. It was found that at 3.44 mM cell growth and ethanol production were significantly affected. However, there was only negligible adverse effect on cell growth and ethanol production at 0.41 mM, which is the expected concentration in bioreactors exposed to syngas. Therefore, it is unnecessary to remove C6H6 from the gas feeding stream. A kinetic model for hydrogenase activity that included inhibition effects of NH4+ and C6H6 was developed. Experimental results showed that NH4+ is a non-competitive inhibitor for hydrogenase activity with KNH4+ of (649 ± 35) mM and KH2 of (0.19 ± 0.1) mM. This KH2 value is consistent with those reported in literature. C6H6 is also a non-competitive inhibitor but a more potent one compared to NH4+ (KC6H6=11.4 ± 1.32 mM). A KH2 value of (0.196 ± 0.022) mM is also comparable with literature and also with the NH4+ study. At a realistic C6H6 concentration of 0.41 mM expected in bioreactors exposed to syngas, hydrogenase activity is expected to be reduced by less than 5%. Forward ADH activity was not adversely affected up to 200 mM [NH4+].From the current work, NH3 should be targeted for removal but it is not necessary to remove C6H6 when designing an efficient gas cleanup system.

Introduction: Short- and long-term health outcomes of children stem from their first 1000 days of development (3 months prior to conception to 2 years postpartum). Research shows a correlation between poor maternal nutrition and adverse birth outcomes. Various factors such as human breast milk (HBM), gut microbiome (GM), and body mass index (BMI) correlate with nutrition. The purpose of this study was to determine if maternal factors such as BMI impact preterm infant microbiome and short chain fatty acid (SCFA) profiles. Methods: Sample Collection: In order to understand the effect of maternal health factors on neonatal GM, deidentified stool samples were collected from the NICU at the MUSC and were utilized for GM and SCFA analysis at ETSU. Microbiome Analysis: GM analysis was performed on stool samples using the Qiagen QIAmp PowerFecal Pro DNA Kit. DNA was sequenced using Amplicon sequence of the 16s rRNA region with a modified Klindworth et al method. GM was analyzed using CLC Genomics Workbench v. 23 where Alpha diversity indexes were calculated with the Abundance Analysis tool and the Beta diversity (inter-sample diversity) was calculated using the weighted Unifrac metric. Short Chain Fatty Acid Analysis: The stool samples were subjected to SCFA extraction and analysis via a modified Schwiertz et al. method. Results: Significance was observed between the groups in microbiome for; C-section (yes, no), gestation (<28, 28-32, 33-36 weeks), week of sample collection (1, 2, 3, 4, >4 weeks), and maternal BMI + antibiotics (no antibiotics + normal, overweight, or obese BMI and antibiotics + normal, overweight, or obese BMI). Significance was detected between the groups in fecal fermentation for; recreational drug use (use, no use), preeclampsia (preeclamptic, not), sepsis evaluation (yes, no), week of sample collection (1, 2, 3, 4, >4 weeks), and Fenton measurements for birth length, birth weight, and occipital frontal circumference (small, average, large for gestational age). Conclusions: These results provide valuable insights into the various maternal and neonatal factors on the GM and SCFA profiles of preterm infants, which can have implications for their overall health and development. It is possible for future adverse health outcomes of premature neonates to be attenuated through HBM ingested and GM.

High gravity (HG) brewing has become the most used strategy for maximizing fermenter productivity in commercial brewing. While HG brewing has many benefits, the additional stress placed on the yeast due to the higher concentration of fermentable sugars in the wort can negatively impact fermentation performance and flavor compound formation. A proper dissolved oxygen (DO) level is vital to guarantee adequate yeast performance during HG fermentations. Dissolved oxygen is vital to yeast viability throughout the fermentation process, as yeast requires oxygen to synthesize vital cell membrane components needed for continued anaerobic growth and cell division. Previous research has demonstrated the importance of DO in wort for regular gravity fermentation and flavor compound production. However, the impact of dissolved oxygen during HG brewing on fermentation performance and how this will impact the production of flavor compounds have not been fully researched. The objectives of this research were to analyze the impact of wort aeration timing and concentration on fermentation performance, flavor stability, and the formation of volatile flavor compounds, determined using gas chromatography. Gas chromatography analysis was modeled after the ASBC Method Beer-48. Flavor stability and staling was analyzed during aging under normal and accelerated conditions utilizing TBA analysis. Pre-pitch oxygen treatments at levels greater than 8 ppm dissolved oxygen significantly increased attenuation when compared to the unoxygenated controls. Post-pitch oxygenation significantly increased attenuation, with DO treatments at levels of 8 ppm showed the most significant decrease in wort specific gravity. Aldehyde, ester, and higher alcohol production were all significantly affected by DO concentration. Aldehyde production decreased with increased DO concentration. Ester production increased from 0 to 8 ppm DO treatment and decreased at DO treatments greater than 8 ppm. Higher alcohol production increased from 0 to 10 ppm and decreased with DO treatments greater than 10 ppm. Greater concentrations v of DO resulted in greater TBA index values after normal and accelerated aging, with accelerated aging producing greater TBA index values than normal aging.

Crude glycerol is the major byproduct of the biodiesel industry. There is an abundance of this byproduct and purifying it for use in industries such as food, pharmaceutical, or cosmetic is prohibitively expensive. Developing an alternative use for crude glycerol is needed. Utilizing it as a carbon source in the fermentation of algae is one potential method for using this under-utilized byproduct. In this research, crude glycerol is used in the mixotrophic production of the alga, Phaeodactylum tricornutum, which is an eicosapentaenoic acid (EPA) producing diatom. Mixotrophic growth is when cells perform autotrophic and heterotrophic modes of growth concurrently. EPA is an omega-3 polyunsaturated fatty acid that has been demonstrated to have a multitude of beneficial health effects, including maintaining human cardiovascular health, treating cancer and human depression diseases, and an anti-obesity effect. In this study, the potential of using crude glycerol in batch mode mixotrophic culture of P. tricornutum was investigated. Once the mixotrophic culture was established, parameters involved in increasing the biomass and EPA production were optimized. These included nitrogen source, level of supplemental carbon dioxide, and concentration of crude glycerol. Using nitrate, 0.08 M crude glycerol, and 3% (vol/vol) carbon dioxide led to the highest biomass productivity of 0.446 g L?? day?? and the highest EPA productivity of 16.9 mg L?? day?? in batch mode culture. The continuous culture of the mixotrophic culture was then performed following the batch culture optimization. The effects of dilution rate were observed in continuous culture with the parameters of nitrate as the nitrogen source, 0.08 M crude glycerol, and 3% (vol/vol) carbon dioxide held constant. The highest biomass productivity of 0.612 g L?? day?? was obtained at D = 0.24 day??. The highest EPA productivity of 16.5 mg L?? day?? was achieved at both D = 0.15 day?? and D = 0.24 day??. The maximum specific growth rate was estimated from the washing out dilution rate and was determined to be around 0.677 day??. Overall, it was found that crude glycerol increases the biomass and EPA productivity of Phaeodactylum tricornutum. Continuous culture with the use of crude glycerol can further increase these measurements. The potential for scaling up studies is demonstrated by these results and can help lead to a market for this abundant, little-used byproduct of the biodiesel industry. / Master of Science

Crude glycerol is a major byproduct if the biodiesel industry. Biodiesel manufacturers are currently facing the challenges of appropriate disposal of this waste material. Crude glycerol is expensive to purify for use in food, cosmetic, and pharmaceutical industries and therefore, alternative methods for use of this crude glycerol are needed. A promising alternative is to use this crude glycerol as a carbon source for microalgae fermentation. In this project, we investigated the use of crude glycerol as a less expensive substrate for the fermentation of the microalgae <i>Schizochytrium limacinum</i> and <i>Pythium irregulare</i> which are prolific producers of omega-3 polyunsaturated fatty acids. Omega-3 fatty acids have many beneficially effects on treating human diseases such as cardiovascular diseases, cancers, and neurological disorders. In addition, the omega-3 fatty acids docosahexaenoic acid (DHA) has been shown to be an important factor in infant brain and eye development. The first part of this study focused on the continuous fermentation of <i>S. limacinum</i>, a prolific producer of DHA. The objective of this study was to examine the algal cellular physiology and maximize its DHA productivity. Two important parameters used in continuous fermentation were studied: dilution rate (D) and feed glycerol concentration (S₀). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L. The cells had a true growth yield of 0.283 g/g, a maximum specific growth rate of 0.692 day⁻¹, and a maintenance coefficient of 0.2216 day⁻¹. The second part of this study focused on morphology issues with <i>P. irregulare</i>, a prolific producer of eicosapentaenoic acid (EPA). <i>P. irregulare</i> has a filamentous morphology, which can make fermentation difficult. The mycelium can stick to the agitation blades resulting in mechanical problems. In addition, this filamentous morphology prevents adequate amounts of oxygen from reaching some cells resulting in decreased productivities. The focus of this research was to control the fermentation conditions to make the algae grow in small pellets, a morphology more suitable for fermentation. In flask culture studies, pellets were formed at an agitation speed of 110 rpm in both regular and baffled flasks. Baffled flasks resulted in pellet formation at 90 and 130 rpm as well. Fermentation studies resulted in pellet formation at agitation speeds of 150 and 300 rpm. Pellets were better able to form when a baffle was not in place. In addition, agitation speed influenced pellet size, with smaller pellets forming at the higher agitation speed. Overall, this study showed that crude glycerol can be used as a carbon source for the continuous fermentation of <i>S. limacinum</i> with high DHA productivity and the morphology of <i>P. irregulare</i> could be controlled by manipulating culture conditions, mainly agitation speed. These results show the potential for scale-up studies for both algal species. / Master of Science

Chronic oxidation linked diseases are on a rise and are one of the leading causes of death globally. Epidemiological evidence increasingly points towards consumption of fruits and vegetables as a preventive way to manage early stages of chronic oxidation linked diseases. Oxidation linked diseases are caused by excessive reactive oxygen species (ROS) generated by a disruption in cellular antioxidant homeostasis due to an overload of calories combined with stress, no excerise and a diet low in antioxidants. Phenolic compounds can not only act as antioxidants but also stimulate the activities of antioxidants enzyme through protective pathways which can help modulate cellular protection. The aim of this dissertation was to use probiotic fermentation to enhance the phenolic and antioxidant compounds in fruit systems which can form the basis of functional food design. The potential of these food systems for disease prevention was investigated in eukaryotic systems through understanding the role of critical metabolic pathways involed in prevention of oxidation linked chronic diseases. Based on structure-function rationale, antioxidant, anti-hyperglycemia and anti-hypertensive potential of phenolic compounds in tea and the effect of extraction time and different degrees of fermentation were investigated in in vitro models. Results indicated that the most fermented teas and a longer extraction time had the highest potential. Further these extracts also had higher H. pylori inhibition potential. Probiotic fermentation of fruit juices with L. helveticus was used to mobilize phenolics and improve biological functionality by maintaining a consistent phytochemical profile. Results indicated that total phenolic and antioxidant potential decreased with feremnetation. However α-glucosidase inhibitory activity and H. pylori inhibitory potential increased with fermentation. Investigation into the mechanism of H. pylori inhibition with fermented cherry extracts revealed inhibition of proline dehydrogenase as the likely mode of action. The potential of fermented apple extracts was further investigated as a phytochemical elicitor in eliciting phenolic and antioxidant response in germinating fava bean. The results indicated a stimulation of phenolic and antioxidant response likely through the stimulation of carbon flux through glycolytic pathways. In yeast, fermented apple extracts accelerated cell death in the presence of peroxide stress in pretreatment model whereas it provided protection against oxidative stress and prevented cell death in concurrent model. Chitosan oligosachharide treatment was investigated as a potential replacement of cancer causing diphenylamine treatment for scald reduction in Cortland apples. Although the treatment did not have any effect on scald reduction, it provides better protection in storage by stimulating phenolic and antioxidant response which related to better health relevant functionality.

Second-generation biofuels have the potential to replace fossil fuels in the energy economy without negatively impacting the food supply. An effective biocatalyst must be able to convert all sugars found in lignocellulosic hydrolysates to biofuels. Few microbes exist in that have both a wide substrate range and high ethanol yields necessary for this process. Mixed culture biotechnology is a promising alternative to the use of single organisms in the production of fuels from lignocellulosic biomass. These systems mimic natural processes for the degradation of lignocellulose and exploit the native capabilities of each microbe. The segregation of metabolic pathways allows for the individual optimization of each step in the process. Preliminary work with a consortium capable of saccharification and fermentation showed promise, but the dynamics were poorly understood. Metabolic modeling is a powerful tool for understanding the interactions between microbes in mixed cultures. The development of accurate models of mixed culture metabolism will help drive the engineering of these systems for industrial applications. In this dissertation, dynamic flux balance analysis is applied to mixed culture systems by combining mathematical reconstructions of pure culture metabolism. By tuning the inoculum to sugar concentration, simulations of Saccharomyces cerevisiae and Escherichia coli mutants engineered to ferment a specific substrate display the potential for improved ethanol production over pure cultures. A framework for translating model predictions to experimental systems was developed for a co-culture of S. cerevisiae and xylose-specific E. coli. The consumption of sugar mixtures was optimized through this method, but the inability of the predicted gains in ethanol production to be replicated in experimental systems reveals the importance of selecting microbes with similar optimal growth conditions. The more compatible microbes S. cerevisiae and Scheffersomyces stipitis were modeled under microaerobic conditions to optimize ethanol production from a mixture of glucose and xylose. To further demonstrate the ability of these systems to ferment lignocellulosic hydrolysates, the effect of furan inhibitors on pure and co-cultures was assessed through modeling and experiment. The work presented here represents the first steps towards engineering and optimizing a microbial consortium for the production of ethanol from lignocellulosic biomass.