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Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable compounds. The goal of this project was to refactor and overexpress b6-f complex proteins in cyanobacteria to improve photosynthesis under dynamic light conditions. Improvement in the photosynthetic system can directly relate to higher yields of valuable compounds such as carotenoids and higher yields of biomass which can be used as energy molecules. Four engineered strains of cyanobacteria were successfully constructed and overexpressed the corresponding four large subunits in the cytochrome b6-f complex. No significant changes were found in cell growth or pigment titer in the modified strains compared to the wild type. The growth assay will be performed at higher and/or dynamic light intensities including natural light conditions for further analysis.
ContributorsNauroth, Benjamin (Author) / Varman, Arul (Thesis director) / Singharoy, Abhishek (Committee member) / Li, Han (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.
ContributorsDufault, Matthew Elijah (Co-author, Co-author) / Wang, Xuan (Thesis director) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Fermentative bioproduction is an efficient production avenue for many small organic acids with less greenhouse gas emissions than petrochemical conversion. Export of these organic acids from the cell is proposed to be mediated by networks of transmembrane transport proteins. However characterization of full transporter networks or the substrate promiscuity of individual transporters is often incomplete. Here, we used a cheminformatic approach to predict previously unknown native activity of E. coli transporters based on substrate promiscuity. Experimental validation in characterized several major putative malate exporters, whereas others were characterized as weak putative lactate exporters. The lactate export network remains incompletely characterized and might be mediated by a large, evolved network of promiscuous transporters.
ContributorsSchneider, Aidan (Author) / Wang, Xuan (Thesis director) / Varman, Arul (Committee member) / Nielsen, David (Committee member) / Department of Finance (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
The increased shift towards environmentalism has brought notable attention to a universal excessive plastic consumption and subsequent plastic overload in landfills. Among these plastics, polyethylene terephthalate, more commonly known as PET, constitutes a large percentage of the waste that ends up in landfills. Material and chemical/thermal methods for recycling are both costly, and inefficient, which necessitates a more sustainable and cheaper alternative. The current study aims at fulfilling that role through genetic engineering of Bacillus subtilis with integration of genes from LCC, Ideonella sakaiensis, and Bacillus subtilis. The plasmid construction was done through restriction cloning. A recombinant plasmid for the expression of LCC was constructed, and transformed into Escherichia coli. Future experiments for this study should include redesigning of primers, with possible combination of signal peptides with genes during construct design, and more advanced assays for effective outcomes.
ContributorsKalscheur, Bethany Ann (Author) / Varman, Arul (Thesis director) / Andino, Jean (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Phenolic polymers such as polyphenols and polyphenylenes are generated industrially for several applications but are typically associated with harsh reaction conditions and environmentally hazardous chemicals, such as formaldehyde. Additionally, hydroxycinnamic acids, such as p-coumaric acid (CA), are found in high concentrations in underutilized lignin-derived hydrolysates and represent a renewable and sustainable feedstock for the production of various aromatics and phenolics. To that end, recently a strain of Corynebacterium glutamicum has been developed by the Joint Bioenergy Institute to express a Phenolic Acid Decarboxylase (PAD), which can convert CA into 4-vinylphenol (4VP). 4VP is cytotoxic but can be polymerized by ligninolytic enzymes such as laccases or peroxidases into less-toxic poly(4-vinylphenol) (PVP). This work investigates the potential of polymerizing 4VP in situ by adding ligninolytic enzymes into the fermentation media to polymerize 4VP into PVP as it is produced, while reducing cellular toxicity to aid in chemical conversion.
The engineered C. glutamicum strain was cultured in the presence of CA to produce 4VP, with a maximum yield of 80.75%. Simultaneously, two ligninolytic enzymes, laccase and horseradish peroxidase (HRP), were explored in an in vitro experiment for their ability to polymerize 4VP, with laccase achieving full polymerization within 45 minutes and HRP able to polymerize 54.06% of 4VP in 24 hours. The resulting polymers were further analyzed by using gas permeation chromatography - nuclear magnetic resonance, validating the synthesis of PVP from 4VP with the addition of laccase or HRP. Finally, the C. glutamicum strain was evaluated for its ability to grow in the presence of hydrogen peroxide, which is a necessary reagent for HRP functionality, and it was able to reach an optical density of 3.69 within 36 hours. These findings suggest that in situ polymerization may be possible. Further work is underway to explore the enzyme kinetics at different pH, validate the potential of polymerization in situ, and study the fermentative benefits associated with in situ polymerization. This will be followed by additional analytical studies to characterize the resulting PVP.
ContributorsEderer, William (Author) / Varman, Arul (Thesis director) / Long, Timothy (Committee member) / Rodriguez, Alberto (Committee member) / Barrett, The Honors College (Contributor) / School of Sustainability (Contributor) / Chemical Engineering Program (Contributor)
Created2022-05
Description
Fossil fuels are currently the main source of energy in the world’s transportation sector. They are also the primary contributor to carbon emissions in the atmosphere, leading to adverse climate effects. The objective of the following research is to increase the yield and efficiency of algal biofuel in order to establish algal-derived fuel as a competitive alternative to predominantly used fossil fuels. Using biofuel commercially will reduce the cost of production and ultimately decrease additional carbon emissions. Experiments were performed using hydrothermal liquefaction (HTL) to determine which catalyst would enhance the algal biocrude oil and result in the highest quality biofuel product, as well as to find the optimal combination of processing temperature and manure co-liquefaction of biomass ratio. For the catalytic upgrading experiments, Micractenium Immerum algae was used in conjunction with pure H2, Pt/C, MO2C, and HZSM-5 catalysts at 350℃ and 400℃, 430 psi, and a 30-minute residence time to investigate the effects of catalyst choice and temperature on the crude oil yield. While all catalysts increased the carbon content of the crude oil, it was found that using HZSM-5 at 350℃ resulted in the greatest overall yield of about 75%. However, the Pt/C catalyst increased the HHV from 34.26 MJ/kg to 43.26 MJ/kg. Cyanidioschyzon merolae (CM) algae and swine manure were utilized in the co-liquefaction experiments, in ratios (algae to swine) of 80:20, 50:50, and 20:80 at temperatures of 300℃ and 330℃. It was found that a ratio of 80:20 at 330℃ produced the highest biocrude oil yield of 29.3%. Although the 80:20 experiments had the greatest biomass conversion and best supported the deacidification of the oil product, the biocrude oil had a HHV of 33.58 MJ/kg, the lowest between the three different ratios. However, all calorific values were relatively close to each other, suggesting that both catalytic upgrading and co-liquefaction can increase the efficiency and economic viability of algal biofuel.
ContributorsMurdock, Tessa A (Author) / Deng, Shuguang (Thesis director) / Varman, Arul (Committee member) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The development of Corynebacterium glutamicum for the microbial production of high-value products has made this bacterium an industrial workhorse. This metabolically engineered microbe is capable of accumulating and secreting flavonoids, a class of high functioning compounds found in plants. In human health, flavonoids are known to have powerful antioxidant, anti-inflammatory, anticancer, and antiviral properties which has led the growing interest to produce these compounds commercially. Recent literature seeks to overcome potential pathway bottlenecks to optimize flavonoid production by regulating protein expression within the central carbon, shikimate, chorismate, and fatty acid synthesis pathways. This paper reviews engineering strategies performed to increase the precursor titers of malonyl-CoA, phenylalanine, and tyrosine for increased flavonoid production.
ContributorsBalbas, Elissa (Author) / Varman, Arul (Thesis director) / Nielsen, David (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Flavonoids are important biomolecules with a variety of pharmaceutical and agricultural applications. Currently, isolating these compounds is done by plant extraction, however this process is hindered by large land and energy requirements. Previous groups have aimed to overcome these challenges by engineering microbes to produce these important compounds, however this is largely bottlenecked by the lack of intercellular malonyl-CoA availability. To remedy this, the genes matB and matC have been identified as coding for malonyl-CoA synthase and a putative dicarboxylate carrier protein, respectively. Other works have successfully engineered two variants, Streptomyces coelicolor and Rhizobium trifolii, of these genes into Escherichia coli, however this has yet to be accomplished in Gram-positive Corynebacterium glutamicum. Additionally, other groups have neglected to attempt tuning these genes with respect to one another by inserting in front of different inducible promoters. This study has successfully assembled two plasmids containing the Streptomyces coelicolor and Rhizobium trifolii variants of both matB and matC. Preliminary fermentations and GCMS results confirmed that little to none naringenin was produced without the matB-matC module. Additionally, preliminary fermentations revealed that the DelAro1 and DelAro3 strains can be used to reduce metabolism of aromatics like naringenin.
ContributorsRonstadt, Jason (Author) / Varman, Arul (Thesis director) / Nielsen, David (Committee member) / Liu, Fang (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2022-05