Chiral amine motifs are found in many bioactive compounds and therefore strategies for their direct asymmetric synthesis are of great interest. Alongside traditional chemical methods, biocatalysis serves as an important tool for the formation of these compounds that can confer the benefits of sustainable catalyst supply and mild reaction conditions. This thesis describes the application of imine reductase (IRED) biocatalysts for the asymmetric reduction of pre-formed imines and the reductive amination of carbonyl compounds to produce chiral amines. These enzymes are relatively recent additions to the toolbox of biocatalysts for chiral amine synthesis and therefore their scope and application is still very much being explored. The research carried out as part of this PhD is presented as a series of manuscripts that have either been published or are planned for submission to peer-reviewed journals. The choice of presenting this thesis in journal format was made because a considerable body of the candidate's PhD research has been published, with the rest planned for publication in the near future. Furthermore, the compiled review articles and research papers lend themselves to a clear thesis narrative and, combined, have taken considerable time and effort to prepare, equal to that of a traditional thesis format. The contents are organised as follows: Chapter 1: an introduction to biocatalysis and its impact on sustainable chemical manufacturing; Chapter 2: a review assessing the current state of the art in imine reductase biocatalysts; Chapter 3: a perspective on the design and implementation of biocatalytic cascades; Chapter 4: a research article on the application of IREDs in a biocatalytic cascade for the synthesis of chiral piperidine and pyrrolidine frameworks; Chapter 5: aims of the PhD project; Chapter 6: a research article on the discovery and investigation of a reductive aminase (RedAm) found within the IRED family; Chapter 7: a research article on the screening of a diverse set of novel IREDs for their ability to facilitate reductive amination; Chapter 8: a research article on the synthesis of complex bulky dibenz[c,e]azepine compounds using IRED and transaminase biocatalysts; Chapter 9: a summary and outlook; Chapter 10: manuscript supporting information further detailing experimental work; Appendix: list of other publications resulting from this doctoral research.

abstract: Trichloroethene (TCE) is a ubiquitous soil and groundwater contaminant. The most common bioremediation approach for TCE relies on the process of reductive dechlorination by Dehalococcoides mccartyi. D. mccartyi use TCE, dichloroethene, and vinyl chloride as electron acceptors and hydrogen as an electron donor. At contaminated sites, reductive dechlorination is typically promoted by adding a fermentable substrate, which is broken down to short chain fatty acids, simple alcohols, and hydrogen. This study explored microbial chain elongation (MCE), instead of fermentation, to promote TCE reductive dechlorination. In MCE, microbes use simple substrates (e.g., acetate, ethanol) to build medium chain fatty acids and also produce hydrogen during this process. Soil microcosm using TCE and acetate and ethanol as MCE substrates were established under anaerobic conditions. In soil microcosms with synthetic groundwater and natural groundwater, ethene was the main product from TCE reductive dechlorination and butyrate and hydrogen were the main products from MCE. Transfer microcosms using TCE and either acetate and ethanol, ethanol, or acetate were also established. The transfers with TCE and ethanol showed the faster rates of reductive dechlorination and produced more elongated products (i.e., hexanoate). The microbial groups enriched in the soil microcosms likely responsible for chain elongation were most similar to Clostridium genus. These investigations showed the potential for synergistic microbial chain elongation and reductive dechlorination of chlorinated ethenes. / Dissertation/Thesis / Masters Thesis Civil, Environmental and Sustainable Engineering 2019

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Around 1990, Arthur proved a local (ordinary) trace formula for real or p-adic connected reductive groups. The local trace formula is a powerful tool in the local harmonic analysis of reductive groups. One of the aims of this thesis is to establish a local twisted trace formula for certain non-connected reductive groups, which is a twisted version of Arthur’s local trace formula. As an application of the local twisted trace formula, we will prove some twisted orthogonality relations, which are generalizations of Arthur’s results about orthogonality relations for tempered elliptic characters. To establish these relations, we will also give a classification of twisted elliptic representations.

Around 1990, Arthur proved a local (ordinary) trace formula for real or p-adic connected reductive groups. The local trace formula is a powerful tool in the local harmonic analysis of reductive groups. One of the aims of this thesis is to establish a local twisted trace formula for certain non-connected reductive groups, which is a twisted version of Arthur’s local trace formula. As an application of the local twisted trace formula, we will prove some twisted orthogonality relations, which are generalizations of Arthur’s results about orthogonality relations for tempered elliptic characters. To establish these relations, we will also give a classification of twisted elliptic representations.

The aim of this study was to investigate the reaction behaviors of nanoscale Fe3O4 and H1/10-[Fe3O4]MgO slurry injection coupled with the electrokinectic (EK) process for remediation of NO3− and Cr6+ in saturated soil. To assure the above-mentioned nanomaterials were capable of reductively adsorbing inorganic pollutants (e.g., NO3− and Cr6+) in the acidic environment in the anode reservoir of the ek remediation system, an investigation on transformation of the concerned nanomaterials in different aqueous solutions (de-ionized water and simulated groundwater ) of different initial pHs (2 and 3.5) was conducted. Due to a high dose of nanoscale Fe3O4 and a resulting serious agglomeration while adsorbing NO3− and Cr6+, the characteristic peaks of the X-ray diffraction (XRD) analysis for nanoscale Fe3O4 remained the same after adsorption experiments. But the situations were quite different in the case of nanoscale H1/10-[Fe3O4]MgO, the characteristic peaks of £\-Fe2O3 in the XRD pattern were detected, confirming that this nanomaterial could reductively adsorb NO3− and Cr6+ in the acidic environment. The effectiveness of using polyacrylic acid (PAA) and soluble starch (SS) to stabilize nanoscale Fe3O4 and H1/10-[Fe3O4]MgO in different aqueous solutions containing humic acid was compared. It was found the former yielded a better stability. Therefore, PAA was chosen to prepare the slurries of target nanomaterials. Then slurry injection coupled with the EK process was tested for remediation of NO3- and Cr6+ in saturated soil. The results showed that the removal efficiency of NO3− was more than 90%, and the NO3− concentration in the anode reservoir was below Taiwan¡¦s Pollution Control Standards of type¢¹Groundwater for NO3−-N. Under the same test conditions, however, the removal efficiency of Cr6+ was unsatisfactory. This might be ascribed to acidification of soil near the anode resulting in high adsorption of Cr2O72− by soil. Thus, a solution to solve this problem has to seeked. The solution lies in how to enhance the contact of the above-mentioned nanomaterials with Cr6+ in the anode reservoir. One possibility is to use the nature of SS would hydrolyze in the acidic environment. Therefore, SS-stabilized nanomaterials in the acidic environment would hydrolyze resulting in the exposure of the soil nanomaterials therein. To this end, SS was used to replace PAA for nanomaterial slurry preparation for remediation of Cr6+. In addition, polarity reversal was practiced in the EK system to maintain a neutral ph of soil and increase the mobility of Cr6+ in soil. Finally, the result showed that nanoscale Fe3O4 and H1/10-[Fe3O4]MgO slurry injection coupled with the polarity reversal electrokinetic system could really enhance the removal efficiency of Cr6+ in the saturated soil. In summary, nanoscale Fe3O4 and H1/10-[Fe3O4]MgO slurry injection coupled with the EK process has been proven to be capable of remedying NO3− and Cr6+ in saturated soil. Meanwhile, the concept of reductive adsorption was realized in this work as well.

The goal of this study was to use molecular biology techniques to access and monitor the efficacy of bioremediation on a trichloroethene (TCE) polluted site. We added emulsified hydrogen releasing materials to stimulate onsite microbial growth and the biodegradation of TCE. This process was known as enhanced bioremediation. In this study, there were two bioremediation sites had been treated anaerobically. Groundwater samples were taken periodically for microbial analysis. Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the variations in microbial community structures during the in situ groundwater remediation. The DGGE DNA bandings were sequenced to determine the 16S rRNA gene sequences and identify the dominate bacterial species. In addition, we used Dehalococcoides spp. 16S rRNA genes as the targets to do real-time PCR. Results show that the emulsified hydrogen releasing materials could enhance anaerobic reductive dechlorination. After addition of emulsified hydrogen releasing materials, we found that the volatile organic compounds concentrations (i.e., TCE, 1, 1-DCE and VC) were decreased. In microbial analysis, the diversities of the microbial community were increased after nutrient supplement. According to the DNA sequencing results, there were 31 bacterial species had been found that related to TCE degradation (i.e., Acidovorax sp., Burkholderiales, Pseudomonas sp., £]-proteobacterium, Comamonadaceae, Iron-reducing bacterium, Hydrogenophilaceae, Clostridium sp., Geobacter sp., Rhodoferax ferrireducens, Dehalospirillum multivorans and Dehalococcoides spp.). Dehalococcoides spp. can be used as a biomarker to evaluate the efficacy of anaerobic bioremediation on a TCE contaminated site. Therefore, we quantified Dehalococcoides populations to explain the capacity of bioremediation after addition of emulsified hydrogen releasing materials to groundwater. Results reveal that Dehalococcoides cell numbers of site A were 4.47¡Ñ103-8.26¡Ñ104 CFU/liter, site B were 4.60¡Ñ102-9.31¡Ñ107 CFU/liter. This data indicated that the addition of emulsified substrate would increase the growth of total Dehalococcoides population under anaerobic conditions. Overall, results from this study demonstrated that the microbial analysis and quantities of Dehalococcoides at different time points can provide useful information to proceed with bioremediation methods.

In this study, a reproducible spectrophotometric method was established and applied to measure reductive capacity of various nanomaterials. Reductive capacity had been implicated in the toxicity of nanomaterials, but a standardized measurement method had been lacking until this work. The reductive capacity of nanoparticles was defined as the mass of iron reduced from Fe3 to Fe2 by unit mass of nanoparticles, in an aqueous solution that initially contained ferric ions. To measure the reductive capacity, the nanomaterials were incubated in a ferric aqueous solution for 16 hours at 37 degrees C, and the reductive capacity of the nanoparticles was determined by measuring the amount of Fe3 reduced to Fe2 using a spectrophotometric method. The reagents 1,10-phenanthroline and hydroquinone were used as a Fe2 indicator and a reducing agent respectively for the assay. To standardize this method, various experiments were carried out. For the initial ferric solution, various Fe salts were tested, and Iron(III) sulfate was chosen as Fe salt for the standard method. The measured reductive capacity of nanoparticles was found to vary with the measurement conditions; the measured reductive capacity increased with increasing the Fe/nanoparticle ratio; the measured reductive capacity increased with incubation time and leveled off after 8 hours of incubation. For hydrophobic materials, the surfactant Tween-20 was added so that the particles could be wetted and suspended in the ferric aqueous solution. After incubation, the particles were removed from the solution by either filtration or centrifugation before applying the spectrophotometric method. In addition, optimal pH and minimum time to reach ultimate color intensity were also found. Carbon-based nanomaterials, standard reference material and metal oxides were measured for their reductive capacities with this method and characterized by transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), x-ray diffraction (XRD), BET measurement and Raman spectroscopy. For some nanoparticles, the reductive capacity was measured for both the pristine form and the form treated by oxidization or grinding. All carbon-based nanomaterials, except for pristine C60, have a significant reductive capacity while reductive capacity of metal oxides is very low. And it was found that reductive capacity can be increased by surface functional groups or structural defects and reduced by oxidization or heating (graphitization). The reductive capacity of a material can play an important role in its toxicology by synergistic toxic effects in the presence of transition metal ions through the Fenton reaction. Moreover, even without transition metal ions, the ability of a material to donate electrons can be involved in toxicity mechanisms via generation of reactive oxygen species.

Abstract The industrial solvents tetrachloroethylene (PCE) and trichloroethylene (TCE) are among the most ubiquitous chlorinated compounds found in groundwater contamination. One potential method for managing PCE/TCE contaminated sites is the intrinsic bioremediation. Recent regulations adopted by U.S. Environmental Protection Agency allow intrinsic bioremediation to be considered as an alternative during development of corrective action plans. In some remediation cases, enhanced bioremediation are performed to accelerate the contaminant biodegradation rate. The main objective of this study was to evaluate the potential of using intrinsic and enhanced bioremediation technologies to clean up PCE/TCE contaminated aquifers. PCE/TCE bioavailability was evaluated by laboratory microcosms under four reduction/oxidation (redox) conditions including aerobic cometabolism, methanogenesis, iron reduction, and reductive dechlorination. Acclimated bacteria, activated sludge, and aquifer sediments from a pentachlorophenol contaminated site were used as the inocula in this study. Methane, toluene, phenol, sludge cake, and cane molasses were used as the primary substrates (carbon sources) in the cometabolism and reductive dechlorination microcosms. Results from this study show that PCE and TCE can be significantly biodegraded under reductive dechlorination and aerobic cometabolism conditions, respectively. All five carbon sources evaluated in this study can be applied as the primary substrates by microbial consortia to enhance the aerobic cometabolism of TCE. The highest TCE degradation rate [Up to 100% of TCE removal (with an initial concentration of 3.6µM)] was observed in the microcosms with toluene enrichment bacteria as the microbial inocula and toluene as the primary substrate. Under reductive dechlorination conditions, both sludge cake and cane molasses could be used as the primary substrates by microbial consortia (from activated sludge and aquifer sediments) and enhanced the biodegradation of PCE. The highest PCE degradation rate [Up to 100% of PCE removal (with an initial concentration of 17µM)] was observed in the microcosms with anaerobic activated sludge as the microbial inocula and sludge cake as the primary substrate. Except for reductive dechlorination microcosms, no significant PCE removal was observed in the microcosms prepared under iron reduction conditions. Results from this feasibility study would be useful in designing a scale-up in situ (e.g., in situ biobarrier system) or on-site bioremediation system (e.g., bioslurry reactor) for field application. Moreover, the application of non-toxic organic waste to enhance PCE/TCE biodegradation has the potential to become an environmentally and economically acceptable technology for the bioremediation of chlorinated-solvent contaminated groundwater.

Perfluorooctane sulfonate (PFOS) and related perfluoroalkyl surfactants (PFAS) are ubiquitous contaminants of increasing public concern due to their environmental persistence, toxicity, and bioaccumulation. These perfluorinated compounds have been used for more than half a century in a wide variety of industrial and consumer products ranging from stain repellents such as Teflon® to aqueous fire-fighting foams and to grease-proof food packing. The public health and environmental risks posed by PFAS have driven environmental agencies and the industry to restrict their use to specific applications where they cannot be replaced by other chemicals. The sources and pathways of PFOS and its derivatives in the environment are not well understood. Analysis of environmental samples is critical to understand the fate, transport and persistence of these emerging contaminants. Techniques based on fluorine nuclear magnetic resonance (¹⁹F NMR) spectroscopy and high performance liquid chromatography (HPLC) with suppressed conductivity detection were successfully developed to monitor the presence of PFAS in water samples. Chromatographic separation of C₄ to C₈ PFAS surfactants was achieved using a C₁₈ reversed-phase column and a mobile phase consisting of a mixture of boric acid and acetonitrile at mixing ratios ranging from 75:25 to 45:55 (v/v). The combination of these two techniques was very effective for characterization and routine quantification of PFOS and related chemicals. Analytical methods based on ¹⁹F NMR, HPLC-suppressed conductivity detection, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were employed to characterize commercial PFOS samples. Linear and branched PFOS isomers in a percentage ratio of 75:25 were identified. Municipal wastewater treatment systems are one of the major sources of PFAS emissions into the environment. The presence of PFAS in sewage sludge from two wastewater treatment plants in Tucson, Arizona, was investigated. Sludge samples were washed with acetic acid and extracted with a mixture of acetic acid and methanol. The extract was cleaned and concentrated by means of solid phase extraction. LC-MS/MS operating in the selective ion monitoring (SIM) mode was employed to assess the presence of perfluorosulfonates, perfluorosulfonamides, and perfluorocarboxylates in sewage sludge samples. PFOS was the only perfluoroalkyl chemical detected in municipal sludge samples at a concentration of 77 ± 5 g kg⁻¹ sludge dry weight. Cost-effective treatment techniques for removing PFAS from industrial effluents are needed to minimize discharges of these pollutants. Reductive dehalogenation is widely applied to the degradation of highly chlorinated compounds. Hence, the susceptibility of PFOS and related compounds to biological and chemical reductive dehalogenation was evaluated in batch assays. PFAS were not reductively dehalogenated by different microbial consortia even after periods of incubation exceeding 2 y, confirming the high resistance of these compounds to microbial degradation. The anaerobic biodegradability of PFOS and perfluorobutane sulfonate (PFBS) samples exposed to electrochemical pretreatment with boron-doped diamond film electrodes was also investigated. The oxidation decreased the concentration of PFAS and dissolved organic carbon in solution, confirming the destruction of these compounds. However, the oxidative treatment did not enhance the susceptibility of PFAS to microbial degradation even after extended periods of incubation (> 1 y). In contrast, PFOS was reductively dehalogenated with a biomimetic system based on vitamin B12 as the catalyst and Ti(III) citrate as the reducing agent. The optimal treatments conditions of the reaction were 260 μM vitamin B₁₂, 36 mM Ti(III) citrate, 70°C and solution pH 9.0. Interestingly, branched PFOS isomers were more prone to degradation by vitamin B₁₂ catalysis compared to the linear isomer. Removal of 3 mol Fper mol of technical PFOS and 12 mol F- per mol of branched PFOS isomers was achieved. Defluorination of PFOS was also observed at environmental relevant conditions of 30°C and pH 7.0, albeit at lower degradation rates. Fluoride and carbon dioxide were identified as the major products of the chemical defluorination. Traces of partially fluorinated volatile compounds were also detected in the headspace. The feasibility of removing PFAS compounds from aqueous streams by sorption onto granular activated carbon (GAC), zeolite, and wastewater treatment sludge was examined in batch isotherm experiments. The fluorocarbon chain and the functional group influenced sorption of the anionic surfactants, PFOS adsorbed more strongly to GAC than perfluorooctanoic acid (PFOA) and PFBS. Activated carbon showed the highest affinity for PFOS (Freundlich K(F) values of 36.7 to 60.9) followed by the hydrophobic, high-silica zeolite NaY (Si/Al 80, K(F) of 31.8) and lastly anaerobic sludge (K(F) of 0.95 to 1.85). GAC sorption is a suitable treatment for the removal of anionic perfluoroalkyl surfactants when present at low concentrations. Fluoride has been identified as the major product of the reductive dehalogenation of PFOS and derivatives. Thus, the toxicity of inorganic fluoride towards the main microbial populations responsible for the removal of organic constituents and nutrients in wastewater treatment processes was also studied. Fluoride concentrations ranging from 18 to 43 mg L⁻¹ caused 50% inhibition (IC₅₀) of the activity of propionate- and butyratedegrading microorganisms and of acetate-utilization by methanogens evaluated under mesophilic and thermophilic conditions. All other microbial populations evaluated in this study, i.e., glucose fermenters, aerobic glucose-degrading heterotrophs, denitrifying bacteria, and H₂-utilizing methanogens tolerated fluoride at very high concentrations (> 500 mg L⁻¹). In the same manner, H₂-utilizing methanogens also tolerated PFOS and PFBS at concentrations as high as 200 and 500 mg L⁻¹, respectively.