R - Teses de Doutoramento / Doctoral Theses
Permanent URI for this collection
Browse
Browsing R - Teses de Doutoramento / Doctoral Theses by advisor "Afonso, Carlos Manuel Magalhães"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
- Chiral pharmaceuticals in the environment : enantiomeric fraction and biodegradation studiesPublication . Ribeiro, Ana Rita Lado Teixeira; Castro, Paula Maria Lima; Tiritan, Maria Elizabeth; Afonso, Carlos Manuel MagalhãesThe importance of the stereochemistry in medicinal chemistry and in pharmacology is well recognized and the diverse behaviour of enantiomers in the presence of chiral entities is fully documented. Therefore biodegradation in biotic media, as occurs in secondary treatment of wastewater treatment plants (WWTPs) can be enantioselective. The significance of chirality in the environment is comparable to the magnitude in medicinal chemistry and pharmacology since enantiomers of chiral contaminants can significantly differ in environmental fate as well as in effects. The need to evaluate the occurrence and biodegradation processes of single enantiomers of chiral pharmaceuticals (CPs) in the environment is imperative by enantiomeric fraction (EF) monitoring of biodegradation processes, wastewaters and surface waters. Assessing EF is only possible through enantioselective validated methods, which are currently scarce. The work described in this thesis comprises the development of new enantioselective analytical methodologies to accurately measure the EFs of CPs in environmental matrices and/or during biodegradation processes, especially for application to monitoring studies and designed biodegradation. The enantioselective analytical methods developed allowed the studies of biodegradation of several CPs in mineral growth medium (MM) inoculated with activated sludge; with the bacterial strain Labrys portucalensis F11; and in wastewater effluents. Additionally an enantioselective method for EFs quantification of multi-residue CPs was developed to monitor effluents of WWTPs and laboratory-scale bioreactors. Thus, this thesis allows increasing the knowledge on EF variation during biodegradation studies and also in environmental matrices. A vancomycin-based chiral stationary phase ChirobioticTM V column (4.6 mm internal diameter (i.d.)) was used to develop and validate two enantioselective analytical methods by High Performance Liquid Chromatography with Fluorescence Detector (HPLC-FD), allowing the analysis of enantiomers of alprenolol (ALP), atenolol (ATE), metoprolol (MET), propranolol (PHO) and fluoxetine (FLX) in biodegradation assays using MM inoculated with activated sludge. Biodegradation of the racemic mixtures was assessed in MM inoculated with activated sludge. The developed HPLC-FD methods under polar ionic mode had several advantages such as a simple sample preparation method; short chromatographic run; low flow rate; no need of solvents for sample preparation or derivatization and the use of an easy to operate, selective and sensitive detector. Biodegradation by activated sludge had slightly higher rates for the (S)-enantiomeric forms of ALP, PHO and MET, with the enhanced biodegradation by acetate as another growth substrate. Biodegradation of ATE and FLX was non-enantioselective. The effect of simultaneous supplementation decreased the biodegradation rates of all enantiomers perhaps due to a competitive mechanism. An enantioselective analytical method by HPLC-FD was optimized and validated using the ChirobioticTM V column (4.6 mm i.d.) to analyse individual enantiomers of FLX and the metabolite norfluoxetine (NFLX). This method preconcetrated 50 mL of wastewater samples through solid phase extraction (SPE) with Oasis® Mixed mode Cation eXchange (MCX) cartridges and allowed the simultaneous analysis of FLX and NFLX enantiomers under reversed mode using an eco-friendly mobile phase constituted by ethanol and ammonium acetate buffer. The method demonstrated to be easy to operate, selective, sensitive and easy to adapt to other matrices. Biodegradation of racemic FLX in microcosms mimicking aquatic environments (WWTP effluents) was followed by the validated method during 46 days with no observation of enantioselectivity in degradation process. The biodegradation of each enantiomer of FLX separately was also evaluated in MM inoculated by the bacterial strain Labrys portucalensis F11. Degradation of the two enantiomers with preferential degradation of the (R)-enantiomer was observed, with no observation of enantiomerization during the monitorization. The metabolite NFLX was not detected during degradation. An enantioselective SPE-HPLC with tandem Mass Spectrometry method was developed with a triple quadrupole mass analyser using the ChirobioticTM V column with 2.1 mm i.d. in reversed mode to quantify enantiomers of seven CPs belonging to different therapeutic classes (ALP, bisoprolol (BSP), MET, PHO, FLX, venlafaxine (VNF) and salbutamol (SBT)) and one metabolite (NFLX). This method used effluent of a laboratory-scale bioreactor for matrix validation and Oasis® MCX cartridges to preconcentrate 250 mL of water samples, being useful for monitoring wastewater effluents and also effluents of laboratory-scale bioreactors. The limits of detection were between 0.65 and 11.5 ng L-1. The use of a selective and sensitive detector allowed the unequivocal identification and quantification of the target enantiomers in complex matrices. The effluents of three municipal WWTPs in Portugal were analysed. Enantiomers of FLX, VNF, BSP, MET and PHO were found. VNF and FLX were found with EFs different from 0.5.
- Microbial degradation of fluorinated compounds : studies on biodegradation mechanisms and biotreatment systemsPublication . Amorim, Catarina Raquel Leite; Castro, Paula Maria Lima; Afonso, Carlos Manuel Magalhães; Carvalho, Maria de Fátima MagalhãesFluoroorganics are an important class of chemicals compound used in a vast number of applications, including pharmaceutical, agricultural and industrial applications, leading to their disposal and accumulation in the environment. Therefore, it is important to understand and explore the biodegradation mechanisms for their removal. The work described in this thesis aimed to investigate the potential of single bacterial strains for biotransformation of fluoroorganics with different complexity as well as to explore their degradation and fate on biofilm bioreactors. In contaminated environments biodegradation can be affected by different factors. In this study, the effect of different parameters on the biodegradation of fluoroanilines (FAs) by Labrys portucalensis F11 was investigated. Strain F11 was able to degrade FAs as sole source of carbon and nitrogen nevertheless supplementation of the culture medium with nitrogen proved to be beneficial for the degradation. Strain F11 was also able to degrade FAs when cells were previously induced for haloaromatic degradation with fluorobenzene (FB). Indeed, higher removal capacity and substrate degradation rates were achieved using FB-induced cells. Co-metabolism of FAs in the presence of FB was also possible but a competitive mechanism between the substrates for the active sites of the enzymes involved in their degradation occurred. A strain capable of aerobically degrading 4-fluorocinnamic acid (4-FCA), strain S2, was isolated from a biofilm bioreactor and identified as belonging to the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as a sole carbon source but the presence of an easy degradable carbon source in the culture medium allowed a faster 4-FCA removal. However, 4-FCA concentrations higher than 1 mM led to a decrease in cell growth rate and in substrate degradation rate, indicating a toxic effect of this compound on S2 cells. A metabolic pathway for the degradation of 4-FCA by strain S2 is proposed on the basis of the identified intermediates. 4-Fluorobenzoate (4-FBA) was the major transient accumulated intermediate and for the first time trans, trans-muconate was reported as an intermediate in a biodegradation pathway. Biological degradation of 4-FCA in rotating biological contactor (RBC) was studied. After development of a stable biofilm, 35 mg L-1 of 4-FCA was intermittently fed to the bioreactor during 2 months, however only limited mineralization occurred. Therefore, the RBC was inoculated with a suspended culture of strain S2 in order to enhance the reactor performance. After bioaugmentation mineralization of 4-FCA was achieved however, a decrease in 4-FCA removal along bioreactor operation was observed. Apart from strain S2, two other 4-FCA degrading strains were isolated from the biofilm. After bioaugmentation, the degraders remained in the bioreactor by the end of the experiment but the intermittent feeding of the target compound probably led to lower cell numbers. Nevertheless, the study reinforced the need of bioaugmentation when dealing with recalcitrant compounds. Fluoroquinolones (FQs) are fluorinated antibiotics, which contamination and persistence in several environmental matrices have been well documented in the past years. Labrys portucalensis was capable to degrade the selected FQs, namely ofloxacin, norfloxacin and ciprofloxacin, when supplied individually or as a mixture, in the presence of an easy degradable carbon source. For concentrations of FQs higher than 3.5 μM an inhibitory effect on growth of the strain was observed. The removal capacity of strain F11 also decreased at high FQ concentration and stoichiometric fluoride release was not achieved. Several intermediates were identified from the degradation of each FQ and the piperazine ring and the fluorine substituent seemed to be the targets of the enzymatic attack. A granular sludge sequencing batch reactor (SBR), established with activated sludge from a wastewater treatment plant (WWTP), was operated for the treatment of an aqueous stream containing FQs. The effects of FQs on different biological processes, such as Chemical Oxygen Demand (COD), nitrogen and phosphorous removal by aerobic granular sludge were investigated. Overall, no significant alterations on COD effluent levels were registered. Nitrifiers present in the granular sludge were not affected by the presence of FQs however the activity of denitrifiers and phosphate accumulating organisms (PAO) seemed to be inhibited. Sorption and desorption of FQs onto aerobic granules occurred. During reactor operation the microbial community dynamics was monitored by plating the biofilm and by 16S DGGE. Shifts in the microbial communities due to FQs exposure were observed, however some bacteria seemed to be resistant to the selective pressure exerted by the antibiotics.