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- Genre: Masters Thesis
Description
DehaloR^2 is a previously characterized, trichloroethene (TCE)-dechlorinating culture and contains bacteria from the known dechlorinating genus, Dehalococcoides. DehaloR^2 was exposed to three anthropogenic contaminants, Triclocarban (TCC), tris(2-chloroethyl) phosphate (TCEP), and 1,1,1-trichloroethane (TCA) and two biogenic-like halogenated compounds, 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP). The effects on TCE dechlorination ability due to 2,6-DBP and 2,6-DCP exposures were also investigated. DehaloR^2 did not dechlorinate TCC or TCEP. After initial exposure to TCA, half of the initial TCA was dechlorinated to 1,1-dichloroethane (DCA), however half of the TCA remained by day 100. Subsequent TCA and TCE re-exposure showed no reductive dechlorination activity for both TCA and TCE by 120 days after the re-exposure. It has been hypothesized that the microbial TCE-dechlorinating ability was developed before TCE became abundant in groundwater. This dechlorinating ability would have existed in the microbial metabolism due to previous exposure to biogenic halogenated compounds. After observing the inability of DehaloR^2 to dechlorinate other anthropogenic compounds, DehaloR^2 was then exposed to two naturally occurring halogenated phenols, 2,6-DBP and 2,6-DCP, in the presence and absence of TCE. DehaloR^2 debrominated 2,6-DBP through the intermediate 2-bromophenol (2-BP) to the end product phenol faster in the presence of TCE. DehaloR^2 dechlorinated 2,6-DCP to 2-CP in the absence of TCE; however, 2,6-DCP dechlorination was incomplete in the presence of TCE. Additionally, when 2,6-DBP was present, complete TCE dechlorination to ethene occurred more quickly than when TCE was present without 2,6-DBP. However, when 2,6-DCP was present, TCE dechlorination to ethene had not completed by day 55. The increased dehalogenation rate of 2,6-DBP and TCE when present together compared to conditions containing only 2,6-DBP or only TCE suggests a possible synergistic relationship between 2,6-DBP and TCE, while the decreased dechlorination rate of 2,6-DCP and TCE when present together compared to conditions containing only 2,6-DCP or only TCE suggests an inhibitory effect.
ContributorsKegerreis, Kylie (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Halden, Rolf U. (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2012
Description
The purpose behind this research was to identify unknown transport proteins involved in lactate export. Lactate bioproduction is an environmentally beneficial alternative to petroleum-based plastic production as it produces less toxic waste byproduct and can rely on microbial degradation of otherwise wasted biomass. Coupled with appropriate product refinement, industrial microbial producers can be genetically engineered to generate quantities of bioplastic approaching 400 million metric tons each year. However, this process is not entirely suitable for large investment, as the fermentative bottlenecks, including product export and homeostasis control, limit production metrics. Previous studies have based their efforts on enhancing cellular machinery, but there remain uncharacterized membrane proteins involved in product export yet to be determined. It has been seen that deletion of known lactate transporters in Escherichia coli resulted in a decrease in lactate production, unlike the expected inhibition of export. This indicates that there exist membrane proteins with the ability to export lactate which may have another similar substrate it primarily transports.To identify these proteins, I constructed a genomic library of all genes in an engineered lactate producing E. coli strain, with known transporter genes deleted, and systematically screened for potential lactate transporter proteins. Plasmids and their isolated proteins were compared utilizing anaerobic plating to identify genes through sanger sequencing. With this method, I identified two proteins, yiaN and ybhL-ybhM, which did not show any significant improvement in lactate production when tested. Attempts were made to improve library diversity, resulting in isopropyl-β-D-1-thiogalactopyranoside induction as a likely factor for increased expression of potential fermentation-associated proteins. A genomic library from Lactobacillus plantarum was constructed and screened for transport proteins which could improve lactate production. Results showed that isolated plasmids contained no notable inserts, indicating that the initial transformation limited diversity. Lastly, I compared the results from genomic screening with overexpression of target transporter genes by computational substrate similarity search. Induced expression of ttdT, citT and dcuA together significantly increased lactate export and thus production metrics as well as cell growth. These positive results indicate an effective means of determining substrate promiscuity in membrane proteins with similar organic acid transport capacity.
ContributorsLee-Kin, Jared (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
Single and double deletion strains of Escherichia coli were grown in paired co-cultures with an intent to identify examples of metabolite exchange and cooperative interactions between strains. The essential genes pheA, argA, tyrA, and trpC, as well as the non- essential genes pykF, pykA, mdh, ppc, and nuoN were deleted from Escherichia coli strains Bw25113 and ATCC 9637. Cultures were paired at three different initial ratios and grown at plate and flask scale. Optical density measurements were used to observe the performance of tested co-cultures, with changes in maximum optical density and growth rate used as indicators of interaction or lack thereof between tested pairs. Auxotrophic strains unable to produce essential amino acids were observed to grow in co-culture but not in monoculture, indicative of metabolite exchange facilitating growth. An increase in optical density for non-essential pairs when compared to the prototrophic parent and precursor monocultures was indicative of metabolite exchange. The initial frequency of paired mutants with non-essential deletions appeared to have an impact on growth performance, but whether this was indicative of any beneficial exchange was not able to be determined from data.
ContributorsFenner, Alexander James (Author) / Nielsen, David (Thesis advisor) / Wang, Xuan (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a
sustainable future. One potential option is the utilization of metabolically engineered
bacteria to produce value-added chemicals during fermentation. Currently, numerous
strains of metabolically engineered Escherichia coli have shown great capacity to
specialize in the production of high titers of a desired chemical. These metabolic systems,
however, are constrained by the biological limits of E. coli itself. During fermentation, E.
coli grows to less than one twentieth of the density that aerobically growing cultures can
reach. I hypothesized that this decrease in growth during fermentation is due to cellular
stress associated with fermentative growth, likely caused by stress related genes. These
genes, including toxin-antitoxin (TA) systems and the rpoS mediated general stress
response, may have an impact on fermentative growth constraints. Through
transcriptional analysis, I identified that the genes pspC and relE are highly expressed in
fermenting strains of both wild type and metabolically engineered E. coli. Fermentation
of toxin gene knockouts of E. coli BW25113 revealed their potential impacts on E. coli
fermentation. The inactivation of ydcB, lar, relE, hipA, yjfE, chpA, ygiU, ygjN, ygfX,
yeeV, yjdO, yjgK and ydcX did not lead to significant changes in cell growth when tested
using sealed tubes under microaerobic conditions. In contrast, inactivation of pspC, yafQ,
yhaV, yfjG and yoeB increased cell growth after 12 hours while inactivation yncN
significantly arrested cell growth in both tube and fermentation tests, thus proving these
toxins’ roles in fermentative growth. Moreover, inactivation of rpoS also significantly hindered the ability of E. coli to ferment, suggesting its important role in E. coli
fermentation
ContributorsHernandez, Michaella (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
Reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and hydroxyl radicals occur naturally as a byproduct of aerobic respiration. To mitigate damages caused by ROS, Escherichia coli employs defenses including two cytosolic superoxide dismutases (SODs), which convert superoxide to hydrogen peroxide. Deletion of both sodA and sodB, the genes coding for the cytosolic SOD enzymes, results in a strain that is unable to grow on minimal medium without amino acid supplementation. Additionally, deletion of both cytosolic SOD enzymes in a background containing the relA1 allele, an inactive version of the relA gene that contributes to activation of stringent response by amino acid starvation, results in a strain that is unable to grow aerobically, even on rich medium. These observations point to a relationship between the stringent response and oxidative stress. To gain insight into this relationship, suppressors were isolated by growing the ∆sodAB relA1 cells aerobically on rich medium, and seven suppressors were further examined to characterize distinct colony sizes and temperature sensitivity phenotypes. In three of these suppressor-containing strains, the relA1 allele was successfully replaced by the wild type relA allele to allow further study in aerobic conditions. None of those three suppressors were found to increase tolerance to exogenous superoxides produced by paraquat, which shows that these mutations only overcome the superoxide buildup that naturally occurs from deletion of SODs. Because each of these suppressors had unique phenotypes, it is likely that they confer tolerance to SOD-dependent superoxide buildup by different mechanisms. Two of these three suppressors have been sent for whole-genome sequencing to identify the location of the suppressor mutation and determine the mechanism by which they confer superoxide tolerance.
ContributorsFlake, Melissa (Author) / Misra, Rajeev (Thesis advisor) / Shah, Dhara (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2024
Description
Directed evolution using genetically diverse libraries is integral to advancing research in industrial microbial production and protein functionality enhancement. This process typically involves a step of sequence diversification and subsequent selection/screening steps for improved variants. While CRISPR-Cas9 systems are known to offer efficient and targeted modification of genes in vivo, concerns arise regarding off-target effects and the emergence of escaper cells evading Cas9 cleavage. This study investigated a strategy to leverage CRISPR-Cas9 counter-selection in Escherichia coli for targeted chromosomal mutagenesis. By designing gRNAs to target a desired region, the spontaneous mutations occurring at the targeted region will potentially disrupt Cas9 binding and thus allow the cell to avoid death caused by Cas9-induced double-stranded DNA breaks. This population of ‘escaper’ cells surviving the counter-selection will have mutations in the gRNA-targeting region at a higher frequency than their non-escaper counterparts. To optimize this counter-selection method, the design for the CRISPR-Cas9 expression system was improved, Cas9 variants with varied fidelities and activities were investigated, and the strategy of using truncated gRNAs for enhanced mutation selectivity was explored. Using the E. coli rpoB gene as a target for editing, the rifampicin-resistant mutation (caused by mutations in rpoB) frequency was increased by more than five orders of magnitude compared to the control E. coli strain without CRISPR targeting. Nanopore DNA sequencing of the mutants’ rpoB region confirmed the promising targeting efficacy of this approach. This study demonstrates a streamlined method for targeted genetic diversification in vivo, facilitating efficient protein engineering in bacterial systems.
ContributorsRick, Rachel Nicole (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2024
Description
Peatlands represent 3% of the earth’s surface but have been estimated to contain up to 30% of all terrestrial soil organic carbon and release an estimated 40% of global atmospheric CH4 emissions. Contributors to the production of CH4 are methanogenic Archaea through a coupled metabolic dependency of end products released by heterotrophic bacteria within the soil in the absence of O2. To better understand how neighboring bacterial communities can influence methanogenesis, the isolation and physiological characterization of two novel isolates, one Methanoarchaeal isolate and one Acidobacterium isolate identified as QU12MR and R28S, respectively, were targeted in this present study. Co-culture growth in varying temperatures of the QU12MR isolate paired with an isolated Clostridium species labeled R32Q and the R28S isolate were also investigated for possible influences in CH4 production. Phylogenetic analysis of strain QU12MR was observed as a member of genus Methanobacterium sharing 98% identity similar to M. arcticum strain M2 and 99% identity similar to M. uliginosum strain P2St. Phylogenetic analysis of strain R28S was associated with genus Acidicapsa from the phylum Acidobacteria, sharing 97% identity to A. acidisoli strain SK-11 and 96% identity similarity to Occallatibacter savannae strain A2-1c. Bacterial co-culture growth and archaeal CH4 production was present in the five temperature ranges tested. However, bacterial growth and archaeal CH4 production was less than what was observed in pure culture analysis after 21 days of incubation.
ContributorsRamirez, Zeni Elizia (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Roberson, Robert (Thesis advisor) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2018
Description
This research explores microbial chain elongation as a pathway for production of complex organic compounds in soils with implication for the carbon cycle. In chain elongation, simple substrates such as ethanol and short chain carboxylates such as acetate can be converted to longer carbon chain carboxylates under anaerobic conditions through cyclic, reverse β oxidation. This pathway elongates the carboxylate by two carbons. The chain elongation process is overall thermodynamically feasible, and microorganisms gain energy through this process. There have been limited insights into the versatility of chain elongating substrates, understanding the chain elongating microbial community, and its importance in sequestering carbon in the soils.
We used ethanol, methanol, butanol, and hydrogen as electron donors and acetate and propionate as electron acceptors to test the occurrence of microbial chain elongation in four soils with different physicochemical properties and microbial communities. Common chain elongation products were the even numbered chains butyrate, caproate, and butanol, the odd numbered carboxylates valerate and heptanoate, along with molecular hydrogen. At a near neutral pH and mesophilic temperature, we observed a stable and sustained production of longer fatty acids along with hydrogen. Microbial community analysis show phylotypes from families such as Clostridiaceae, Bacillaceae, and Ruminococcaceae in all tested conditions. Through chain elongation, the products formed are less biodegradable. They may undergo transformations and end up as organic carbon, decreasing the greenhouse gas emissions, thus, making this process important to study.
We used ethanol, methanol, butanol, and hydrogen as electron donors and acetate and propionate as electron acceptors to test the occurrence of microbial chain elongation in four soils with different physicochemical properties and microbial communities. Common chain elongation products were the even numbered chains butyrate, caproate, and butanol, the odd numbered carboxylates valerate and heptanoate, along with molecular hydrogen. At a near neutral pH and mesophilic temperature, we observed a stable and sustained production of longer fatty acids along with hydrogen. Microbial community analysis show phylotypes from families such as Clostridiaceae, Bacillaceae, and Ruminococcaceae in all tested conditions. Through chain elongation, the products formed are less biodegradable. They may undergo transformations and end up as organic carbon, decreasing the greenhouse gas emissions, thus, making this process important to study.
ContributorsJoshi, Sayalee (Author) / Delgado, Anca G (Thesis advisor) / Torres, César I (Committee member) / van Paassen, Leon (Committee member) / Arizona State University (Publisher)
Created2018
Description
The application of microalgal biofilms in wastewater treatment has great advantages such as abolishing the need for energy intensive aerators and recovering nutrients as energy, thus reducing the energy requirement of wastewater treatment several-fold. A 162 cm2 algal biofilm reactor with good wastewater treatment performance and a regular harvesting procedure was studied at lab scale to gain an understanding of effectual parameters such as hydraulic retention time (HRT; 2.6 and 1.3 hrs), liquid level (LL; 0.5 and 1.0 cm), and solids retention time (SRT; 3 and 1.5 wks). A revised synthetic wastewater “Syntho 3.7” was used as a surrogate of domestic primary effluent for nutrient concentration consistency in the feed lines. In the base case (2.6 hr HRT, 0.5 cm LL, and 3 wk SRT), percent removals of 69 ± 2 for total nitrogen (TN), 54 ± 21 for total phosphorous (TP), and 60 ± 7 for chemical oxygen demand (COD) were achieved and 4.0 ± 1.6 g/m2/d dry biomass was produced. A diffusion limitation was encountered when increasing the liquid level, while the potential to further decrease the HRT remains. Nonlinear growth kinetics was observed in comparing SRT variations, and promoting autotrophic growth seems possible. Future work will look towards producing a mathematical model and further testing the aptness of this system for large-scale implementation.
ContributorsHalloum, Ibrahim (Author) / Torres, César I (Thesis advisor) / Popat, Sudeep C (Committee member) / Rittmann, Bruce E. (Committee member) / Arizona State University (Publisher)
Created2016
Description
Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
ContributorsBlake, Mellecha (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016