Browsing by Subject "Biodegradation"

Now showing 1 - 7 of 7
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    Adaptive Expansion of Biodegradation by Pseudomonas Putida F1
    (2017-06) Chan, Bao
    Pseudomonas putida F1 (PpF1) catabolizes aromatic compounds via benzoate dioxygenase, phenylacetyl-CoA epoxidase, p-cymene-monooxygenase, and toluene dioxygenase-mediated pathways, and the latter is shown here to be highly flexible, supporting growth on previously untested aromatic alkenes, esters, amides, alcohols, amines, and multi-ring compounds. P. putida F1 is a highly studied model aromatic hydrocarbon oxidizer that grows on relatively few mono-substituted benzenes despite its genome encoding a toluene dioxygenase enzyme that oxidizes more than 150 compounds. While toluene dioxygenase and toluene dihydrodiol dehydrogenase oxidize many mono-substituted benzene ring compounds, further enzymatic processing may be inhibited, leading to accumulation of the respective catechol or 2-hydroxy-6-oxo-2,4-dienoate intermediate. The present study demonstrated that P. putida F1 can undergo adaption leading to a more expanded growth range of mono-substituted benzene ring compounds than had been previously demonstrated. Studies with well-characterized mutant derivatives of P. putida F1 and growth on expected metabolites of the toluene dioxygenase pathway indicated that the newly described metabolism was dependent on the Tod pathway enzymes. This study also revealed the interplay between the Tod pathway enzymes and catabolism of the aromatic acids liberated by TodF. TodABCDEF processing of allylbenzene and 1-phenylethanol liberates 3-butenoic acid and lactic acid, respectively, both of which support growth of P. putida F1.
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    Effects of Organic Carbon on the Biodegradation of Estrone in Multiple Substrate, Mixed-Culture Systems
    (2014-08) Tan, Tat Ui
    This dissertation describes the study of the effect of organic carbon on the biodegradation of estrone (E1) in multiple substrate, mixed-culture systems. In exploring this topic, important degradation mechanisms related to organic carbon were tested to determine which, if any, play an important role. Additionally, the effects of organic carbon concentrations, loads, and quality on E1 degrading activity of cultures from a wastewater treatment system were determined. Catabolic repression effects on E1 degradation was studied by adding synthetic septage to an E1 degrading culture to determine if degradation rates were affected. No differences in first-order E1 degradation rates between test and control reactors were observed in the 2 h or 8 h period following the addition of synthetic septage, ruling out catabolic repression as an important mechanism in E1 degradation in wastewater treatment-like conditions. Cultures were grown in membrane bioreactors (MBRs) with and without exposure to E1 to determine if (i) E1 exposure is necessary for E1 degrading ability, and if so (ii) whether multiple substrate utilization and/or cometabolism play an important role in the degradation of E1. These cultures were capable of degrading E1 regardless of prior exposure. Higher rates of E1 degradation were observed in cultures with prior E1 exposure, and a lag phase of 6 h was observed in cultures without prior E1 exposure. These results indicate that E1 was degraded metabolically, demonstrating that multiple substrate utilization is the key mechanism for E1 degradation. Longer term effects of organic carbon concentrations on E1 degrading activity were explored by comparing cultures operating under starvation conditions and cultures operating on a daily feeding cycle. Cultures fed daily showed a large initial increase in E1 degradation activity, attributable to a corresponding increase in biomass. Subsequently, however, E1 degradation activity dropped substantially even though biomass continued to increase, suggesting that E1 degraders were outcompeted when subjected to repeated exposure to high organic carbon concentrations. Conversely, starvation cultures had moderate but sustained increases in E1 degradation rates. Another experiment using MBRs to distinguish organic loads from organic concentrations confirmed the positive effect of organic carbon loads on E1 degradation via biomass growth, indicating that high organic carbon concentrations rather than loads were responsible for the drop in E1 degradation rates. A follow-up study was carried out to determine if altering the duration between feeding cycles could mitigate the negative effects of high organic carbon concentrations on E1 degradation. When cultures were exposed to high organic carbon concentrations (600 mg COD/L over a 6 d period), increasing the duration between feeding cycles improved performance. Conversely, at lower organic carbon concentrations (180 mg COD/L over a 6 d period), no differences in E1 degrading activity was observed. Effects of organic carbon quality on E1 degradation were explored using aged synthetic septage and waters from various treatment and natural sources to culture mixed communities. In these experiments, spectrophotometric methods (specific UV absorbance, spectral slope ratios, excitation-emission matrices, and fluorescence index) were used to characterize organic carbon. Additional analyses and experiments were conducted to rule out organic carbon, nitrogen species, and trace element concentrations as complicating factors. These experiments showed that microbially-derived organic carbon was associated with E1 degrading ability, while organic carbon from natural water sources (river and lake) was not. Furthermore, the experiments with aged synthetic septage suggest that products from cell lysis and/or microbial products under stress by starvation may be important for E1 degradation. Overall, this work shows that multiple substrate utilizing bacteria are important for E1 degradation in wastewater treatment-like systems and indicates various organic carbon parameters that are vital for the selection of these bacteria.
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    Improved prediction of biodegradation pathways: visualization and performance.
    (2011-02) Gao, Junfeng
    The University of Minnesota Pathway Prediction System (UM-PPS) (http://umbbd.msi.umn.edu/predict/) is a rule-based system that predicts plausible pathways for microbial degradation of organic compounds. Its biotransformation rules are based on reactions found in the University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.msi.umn.edu/) or in the scientific literature. Since the UM-PPS was created in 2002, its rule base has grown to 275 entries. The original system predicted one level of prediction at a time. It provided a limited view of prediction results and heavily relied on manual interventions. It matched the query compound with all biotransformation rules one by one, which was a time-consuming process. In 2008, the two-level visualization was first implemented to allow users to view two levels of predictions at a time. However, this visualization approach was usually not able to show the complete metabolism of a query compound, and users still needed expert knowledge to make educated choices to continue the prediction. In 2009, we started to develop a multi-level visualization and, simultaneously, work on increasing prediction speed. In 2010, the multi-level visualization was implemented to predict up to six levels of predictions at a time. Not only more products, but also common intermediates and cleavage products are displayed. Users can view prediction alternatives much more easily in a tree-like interactive graph. A multi-level prediction can be computationally intensive and requires users to wait longer than desired for the prediction results. Therefore, we used a multi-thread computing strategy that decreased the prediction run-time by half. We balanced the computing threads and pre-loaded all UM-PPS database tables to permit quick access to its data. Both of these improvements resulted in an additional 30% decrease in prediction run-time. We conducted a simulation study and used another web server to reduce the queuing interference by over 85%. Beta testers were satisfied with its visualization and performance. The above improvements lead to a smarter and faster UM-PPS that has continued its growth in the past 4 years. It now displays better graphical results and predicts biodegradation pathway in a timely manner.
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    Isolation of Lignin-converting Microbes Contributing Towards Recalcitrant Carbon Degradation in Boreal Forest System
    (2022-08) Singh, Nandini
    Plant biomass, composed of cellulose, hemicellulose and lignin, are being explored as renewable carbon feedstock that could replace a significant amount of petroleum-derived chemicals and other products. Lignin, a component of wood, is the second most abundant natural organic polymer after cellulose. It provides strength and rigidity to plants and is highly recalcitrant to degradation due to its complex, three-dimensional structure. The valorization of lignin is essential for viable and sustainable uses of lignocellulosic biomass for the production of renewable fuels and chemicals. In order to achieve this, microbial degradation is extensively researched as microorganisms have evolved different enzymatic and/or non-enzymatic strategies to utilize biomass (Janusz et al., 2017). In boreal forest ecosystem, brown rot fungi are dominant players for decomposing and recycling carbon sources sequestered in tree biomass, but the carbohydrate-selective nutritional mode of these fungi does not allow them to consume all the forms of carbon in wood, with the undecayed lignin residues creating a recalcitrant carbon pool in the forest ecosystem. In this research, we investigated up to 200 functional microbes, including fungi and bacteria, involved in the breakdown of lignin components in this distinctive brown rot niche. Preliminary screening of the microbes was performed using indicator dyes, resulting in eight fungi and fourteen bacteria which were then screened to be further characterized. These isolates were identified using DNA extraction, PCR and sequencing and were further evaluated for their ability to degrade and metabolize lignin and aromatic lignin monomers. We anticipate understanding the lignin-degrading characteristics of microbes occurring naturally and its role in industrial lignin conversion or bioremediation of related recalcitrant aromatic hydrocarbons.
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    Plastic Biotransformation Technologies: Development of a Novel Environmental QPCR Assay for Polyethylene Terephthalate Hydrolase, and Isolation/Characterization of Polyethylene Degrading Fungi and Bacteria from Environmental Samples
    (2020-08) Wedin, Nelson
    Plastic production, use, and accumulation in the environment—including in the bodies of humans and other animals—have been increasing for decades and are a cause of growing global concern. Common plastic waste is generally considered to be non-biodegradable. In recent years, though, a growing assortment of bacteria and fungi capable of degrading a variety of common recalcitrant plastics have been identified. In general, the enzymes capable of depolymerizing long-chain hydrophobic plastic polymers are not well studied. However, Poly(ethylene) Terephthalate (PET) Hydrolase is well described in the literature and is thus a suitable target for molecular identification and quantification technologies. PET is the plastic polymer used in most plastic water bottles and in polyester fabric. The discovery of PET-degrading organisms and PET hydrolases is leading to the generation of biochemical technologies for the recycling and upcycling of PET, as well as the search for PET hydrolases that have greater activity on commercially relevant PET polymers. The incidence of PET hydrolase in metagenomes appears rare, though the quantification of PET hydrolases in environmental samples is unknown. Because plastic-biotransforming organisms are considered rare and slow growing, the process of isolating and characterizing these organisms is long and involved. This thesis presents two distinct, but interrelated, experimental trajectories related to the advancement of the study of plastic biotransformation. The first study focused on the molecular level, and the second study focused on microorganisms. In the first study, novel Quantitative Polymerase Chain Reaction (QPCR) primers were developed and tested for the ability to selectively amplify PET hydrolase genes from environmental samples. The products from these primers, used on eight environmental DNA extracts, were subjected to amplicon sequencing. Multiple sequence analysis methods confirmed the successful amplification of published PET hydrolase sequences, as well as sequences that show a high potential for being PET hydrolases. The on-target hit percentage and on-target hits varied substantially across samples, and this assay will require further optimization for specificity and quantification efficacy before it can be used for absolute quantification (i.e., gene copies/ ng DNA). There is reason to suggest that this assay can measure relative abundance of PET hydrolases, and thus relative genetic PET bioconversion potential. By providing comparative analysis that is both faster and less expensive than traditional techniques, this tool enables the rapid determination of ideal conditions to find and cultivate PET hydrolytic organisms. The core results of this analysis are presented in Figures 26, 28, and 29. In the second study, the focus was to enrich for and isolate (as individual species or consortia), identify, and evaluate microorganisms capable of Polyethylene (PE) biodegradation and biomineralization by culturing microbes in media where PE is the sole carbon source. Although the impact on the environment of PET (the polymer studied in the first study) is substantial, it pales in comparison to the impact of PE, which is used primarily for single-use items and is the most abundant type of plastic manufactured on the planet. Currently, no enzymes capable of degrading PE are well described, though some fungi and bacteria have been shown to degrade and utilize PE as a carbon source. In this set of experiments, preferential focus was given to fungi. Microbes that degrade and live on LDPE powder were enriched from environmental sources. A cogent argument for the confirmation of Low-Density PE (LDPE)-biodegrading organisms is presented from the limited data available (see below for limitations resulting from COVID-19 lockdown). LDPE-biodegradation can be seen in the isolate “Ath” (flask 6, a filamentous fungi that macroscopically appears to be Trichoderma sp.). The macroscopic observation of PE biotransformation for culture “Ath” is documented in Figure 33, where Flask 6 clearly shows modification to the PE powder. Modification increases with longer incubation and is not observed in the otherwise-identical non-inoculated control (Flask 34, Figure 33). Similar results are observed for other cultures, along with the growth of biomass and spore production. Thus, LDPE-biodegradation is also the most likely explanation for at least nine other environmental isolates. And microscopic confirmation of growth in this culture as well as others is presented in conditions where the only carbon source is PE powder. Both bacteria and fungi were shown to degrade the low molecular weight PE powder, though quantitative analysis on commercially relevant PE films was not completed. Tentative taxonomic hypotheses and the exciting possible implications of PE degradation within these taxa are presented, though genetic identification was also unable to be performed due to lockdowns. This research project was cut short prematurely due to mandatory laboratory lockdown in response to the COVID-19 pandemic. While both prongs of the studies described in this thesis were affected, the isolation and PE biodegradation assay was more seriously limited in that all quantitative analyses were unable to be performed. The discussion section reflects the limitations that resulted, as well as the adjustments that were made to compensate for these limitations.
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    Structural and mechanistic studies of cyanuric acid hydrolase and biodegradation of 1,3,5-triazine
    (2015-10) Cho, Seunghee
    Microorganisms have developed the greatest diversity of enzymatic pathways to allow their successful proliferation in the natural world. Those enzymatic pathways are the result of interactions between microorganisms and their habitat leading to adaptations by the microorganisms. As knowledge about the enzymatic pathways accumulates, increasingly those pathways can be used to degrade recalcitrant pollutants. The biodegradation pathway of s-triazine compounds is an example. Cyanuric acid hydrolase (CAH) is the one of the enzymes in the s-triazine degradation pathway. CAH is of particular interest because it hydrolyses cyanuric acid that is the by-product of tri-chlorocyanuric acid decomposition which has been used in swimming pool disinfection. The first three chapters will deal with the mechanism of CAH mainly using X-ray crystallography. Chapter 2 describes the structural characteristics of the active site of CAH with emphasis on emerging evidence that a serine in domain B is the nucleophilic serine in the catalytic cycle. Evidence from mutational studies, Burgi-Dunitz angle analysis, and sequence alignments all support the hypothesis that the serine in domain B is the nucleophilic serine. Direct evidence is presented in chapter 3 for this serine acting as the nucleophile by our solving an X-ray structure showing a reaction intermediate covalently attached to this serine (the so-called “acyl intermediate”). The X-ray structure also shows 24 monomers in the crystal’s asymmetric unit and different active sites within the asymmetric unit contain different stages of the reaction such as the unreacted substrate, the acyl intermediate, carboxybiuret, biuret, and an empty active that has expelled the product. The thesis concludes with chapter 5 that provides the novel biodegradation pathway of unsubstituted triazine (1,3,5-triazine) by Acinetobacter sp. Trz. Although there have been a lot of studies on substituted triazines biodegradation, no study on unsubstituted triazine (1,3,5-triazine) has been done previously. The chapter 5 describes the isolation of Acinetobacter sp. Trz that utilizes 1,3,5-triazine as its sole nitrogen source and the novel biodegradation pathway of 1,3,5-triazine. The 1H-NMR and HPLC analysis shows that 1,3,5-triazine first transforms to formamidine (FAD), formamide (FD) and ammonia non-enzymatically and then enzymes likely trasnform formamidine to formamide and formamide to formate and ammonia.
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    Sustainable aliphatic polyester material design
    (2021-08) Batiste, Derek
    Plastics are ubiquitous materials that are primarily made out of synthetic polymers. There are many global problems that stem from the way societies produce, use, and discard plastic materials. A majority of synthetic polymers are synthesized from finite fossil fuel feedstocks. Waste management infrastructures have failed to coral large portions of discarded plastics, resulting in their proliferation in natural environments. Recycling technologies are severely limited due to problems from the collection and sorting infrastructures and the reduced mechanical performance of most reprocessed materials. Sustainable material design is one of many approaches which seeks to invent and discover new materials, processes, and technologies that can help remediate these issues. This dissertation presents several projects—each motivated by one or more subcategories defined in the NSF Center for Sustainable Polymers’ “Sustainable Polymer Framework”— that explore various aspects of aliphatic polyesters as sustainable materials. Chapter 1 provides historical perspectives on the rise of synthetic plastic use and the shifting value of sustainable development. Chapter 2 focuses on the metal-free polymerization of methyl-ε-caprolactone (MCL) isomers using the organocatalysts diphenyl phosphate and dimethyl phosphate. Chapter 3 focuses on the use of poly(methyl-ε-caprolactone) (PMCL) to synthesize mechanically strong thermoplastic poly(urethane-urea)s and chemically crosslinked polyurethanes. Additionally, chemical recyclability of these materials is explored through monomer recovery experiments with Lewis acid catalysts. Chapter 4 comprises multiple studies assessing the end-of-life of PMCL-based materials and its hydrolysis products. The cytotoxicity of PMCL-based elastomers and the hydrolysis products are explored as well as the degradation of the elastomer materials in industrial composting conditions. The biodegradation of PMCL is explored in further detail through the study of isotopomers of the hydrolysis product in natural soil incubation studies.