ESB - Teses de Doutoramento / Doctoral Theses
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Browsing ESB - Teses de Doutoramento / Doctoral Theses by Field of Science and Technology (FOS) "Engenharia e Tecnologia::Engenharia do Ambiente"
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- Aerobic granular sludge as a source of extracellular polymeric substances and the potential of the technology combined with bioaugmentation to treat industrial wastewaterPublication . Oliveira, Ana Sofia Teixeira; Castro, Paula Maria Lima e; Amorim, Catarina Raquel Leite; van Loosdrecht, MarkA variety of organic pollutants reach wastewater treatment plants (WWTP), often associated with high salinity levels, making their treatment challenging. Aerobic granular sludge (AGS) technology is thought to protect the microbial communities from stress due to the high content of extracellular polymeric substances (EPS). There is an increasing need to turn WWTP more efficient, with a range of opportunities for resource recovery to integrate them into the circular economy concept. The work described in this thesis aimed to explore AGS biomass as a source of EPS and to understand the variations of EPS production facing different stressors, namely 2-fluorophenol (2-FP) and salinity. Particular attention was given to the microbial communities, diversity and function, of the investigated systems. The recovery of EPS from AGS represents an opportunity for valorization of surplus biomass. AGS from a full-scale WWTP treating urban wastewater was regularly collected for 4 months to assess variability in EPS composition and in granular morphology. Variations in the EPS composition occurred with time, with proteins and humic acids as the main EPS components and polysaccharides and DNA as minor constituents. An extra purification step led to the recovery of a purer EPS form with a rather homogeneous composition however the yield of each EPS component decreased, especially for polysaccharides. Yield and product homogeneity are key features for downstream application of the recovered EPS. The effect of intermittent short-term loadings of 2-FP and low to moderate salinity wastewater on the performance and EPS production of an AGS system was studied. Ammonium removal was highly inhibited by stressors, recovering when 2-FP feeding ceased. Phosphate removal, initially disturbed by exposure to stress conditions, recovered when stressors were still present. EPS composition and concentration in the granules decreased from 133.3 to 33.7 mg/g VSS of AGS during the first phases of stress but its production recovered to 176.1 mg/g VSS of AGS even in the stressor’s presence. The nutrient removal recovery after exposure to stressors and the increased EPS production response support the robustness of AGS systems to deal with intermittent stressful conditions. EPS recovered from AGS were used as an immobilizing agent for Rhodococcus sp. FP1, a 2-FP degrading strain. The produced EPS granules exhibited 2-FP degrading ability of 100%, retaining its original activity up to 2 months storage. Moreover, the EPS granules were used to bioaugment an AGS reactor intermittently fed with low to moderate saline wastewater amended with 2-FP. After bioaugmentation, complete 2-FP removal occurred and phosphate and ammonium removal (previously impaired by 2-FP load) improved from 14 to 46% and from 25 to 42%, respectively. After bioaugmentation, strain FP1 was detected up to 3 days in the reactor effluent by qPCR and eleven bacterial isolates able to degrade 2-FP were retrieved from the AGS. Maintenance of cell viability through storage and improvement of bioreactor.
- Fluoroquinolone antibiotics in the environment : biodegradation studies and presence in several environmental matricesPublication . Maia, Alexandra Sofia Moreira Amendolia da Costa; Castro, Paula Maria Lima; Tiritan, Maria Elizabeth; Cass, Quezia BezerraThe use of fluoroquinolone antibiotics for therapeutic, prophylactic and growth promotion applications is increasing worldwide. Due to its halogenated characteristics, ecotoxicity and persistence, a special importance is given to their occurrence in the environment. Since some fluoroquinolones have one or more stereogenic centers, they give rise to pairs of enantiomers. The pharmacological properties and toxicity of enantiomers can be very different. The stereochemistry of these compounds represents a major challenge in terms of its environmental monitoring and is frequently ignored. The presence of fluoroquinolones has been described in many environmental matrices, but few studies were done in Portugal regarding environmental monitoring studies. Degradative processes for the removal of fluoroquinolones have also been subject of several studies, but none has considered the relevance of the stereochemistry. The work described in this thesis comprises the development of new analytical methods using liquid chromatography with fluorescence detection (LC-FD) to assess biodegradation of chiral and achiral fluoroquinolone antibiotics and liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify fluoroquinolones residues in different environmental matrices, such as influents and effluents of waste water treatment plants. As the metabolites/degradation products originated during degradation processes may present higher ecotoxicity than the parent compound this work also aimed to identify the most important by-products originated during these processes. Fluoroquinolones by-products formed during degradation assays were identified using an integrated analytical approach and these studies enabled the proposal of the chemical structures of two new metabolites for ofloxacin and norfloxacin, never reported before. Biodegradation of chiral fluoroquinolone antibiotics in laboratory experiments using activated sludge and two single bacterial strains were followed by the enantioselective methods developed. An analytical method for quantification and achiral assessment of fluoroquinolones in environmental matrices was developed and applied to wastewater treatment plants influents and effluents. A ristocetin A-based chiral stationary phase (Chirobiotic™ R column) was used for the development of an enantioselective method by Liquid Chromatography with Fluorescence Detection, which after validation allowed for the analysis of ofloxacin and levofloxacin enantiomers in biodegradation assays performed with activated sludge. Biodegradation of ofloxacin proved to be enantioselective and both fluoroquinolones biodegradation extents were enhanced with supplementation with acetate. Subsequently, mass spectrometric analysis confirmed the racemization of the pure enantiomer levofloxacin during the biodegradation by the activated sludge inoculum. These results were reported for the first time in this work. Two single bacterial strains (Labrys portucalensis F11 and Rhodococcus sp. FP1) were assessed regarding their enantioselective behavior on the biodegradation of ofloxacin and levofloxacin at a laboratory-scale microcosm, using the previously validated analytical method. Biodegradation studies were conducted under different initial concentrations of the two fluoroquinolones and different supplementations with acetate were tested. Racemic ofloxacin was enantioselectively biodegraded by both bacterial strains, with the S-enantiomer showing preferential removal and consequently enrichment of the R-enantiomer was observed. Both fluoroquinolones were degraded to an almost complete extent by strain F11. Racemization of levofloxacin was observed in its biodegradation by strain F11 and confirmed using mass spectrometric analysis. This work described enantioselective biodegradation of ofloxacin and levofloxacin by single bacteria for the first time. A solid phase extraction – liquid chromatography with tandem mass spectrometry method was developed with a triple quadrupole mass analyzer for the quantification of seven fluoroquinolone antibiotics in environmental aqueous matrices. Oasis® HLB columns were used for the clean-up and concentration of 100 mL influent and effluent samples from fifteen wastewater treatment plants. Method detection limits ranged from 6.7 to 59.0 ng L-1. High concentrations of ofloxacin, up to 4587.0 ng L-1, were found in influent samples. Ofloxacin and ciprofloxacin were the fluoroquinolones most frequently detected. Norfloxacin and prulifloxacin were not found in any of the analyzed samples.
- Utilization of plant growth-promoting bacteria to ameliorate iron nutrition in legumesPublication . Costa, Mariana Roriz Lemos; Vasconcelos, Marta Wilton Pereira Leite de; Carvalho, Susana Maria Pinto de; Castro, Paula Maria Lima eIron (Fe) deficiency is an important micronutrient disorder that leads to severe yield losses and low nutritional crop value, particularly in calcareous soils. There is an urgent need to find sustainable and greener agricultural practices to achieve higher crop yields with higher nutritional value. Biofortification allows the increase of micronutrient concentrations in edible crop tissues and contributes to achieving such demands. The utilization of bioinoculants (BIs) with plant growth-promoting bacteria (PGPB) has been suggested as a promising approach for biofortification and prevention of Fe deficiency. So far, little work has been done on the role of PGPB in soybean (Glycine max) grown under alkaline conditions. The main purpose of this study was to test the potential of PGPB on plant growth and Fe uptake, unveiling mechanisms underlying Fe uptake and accumulation. Soybean was selected as a model species since it is severely affected by Fe deficiency and several underlying factors related to Fe homeostasis are identified. Firstly, an in-depth and critical literature review was conducted concerning the global importance of Fe and the Fe deficiency, the importance of soybean in the fulfillment of global policies, and the potential of PGPB as a sustainable approach to improve Fe nutrition and cope with Fe deficiency. Concerning the experimental work, the first study of this thesis aimed to evaluate the ability of 24 PGPB strains from a CBQF collection to enhance Fe uptake-related processes in soybean grown for 21 days in calcareous soil. Sphingobium fuliginis ZR 1-6 and Pseudomonas jessenii ZR 3-8 were selected based on their in vitro ability to produce indole-3-acetic acid (IAA), 1-aminocyclopropane-1- carboxylic acid (ACC) deaminase, siderophores, and organic acids, to tolerate high pH, and to reduce Fe3+. Bacterial isolates were inoculated singly and as a mixture, and a series of morphological, physiological, and molecular parameters were evaluated. S. fuliginis improved ferric chelate reductase (FC-R) activity (111 %), FRO2 expression (646 %), and root Fe (62 %); combined inoculation fostered Fe accumulation in trifoliates (144 %) and increased IRT1 (239 %) and FER4 expression (5036 %). Overall, S. fuliginis alone or in combination with P. jessenni were the best treatments. In a second study, PGPB were isolated from root tissues and rhizosphere of soybean grown in a Portuguese soil; 76 bacterial strains were isolated from roots (53 %), rhizosphere (29 %), and shoots (18 %), and 29 genera were identified. Two bacterial strains – B. licheniformis P2.3 and B. aerius S2.14 – were selected for in vivo experiments, and inoculated plants were grown to maturity. Photosynthetic parameters, chlorophyll content, total fresh weight, and Fe concentrations were not significantly affected by inoculation. Nevertheless, inoculation with B. licheniformis increased pod number (33 %), decreased FC-R activity (45 %), and increased expression of Fe-related genes; inoculation with B. aerius decreased root length (20 %), FC-R activity (55 %), and FRO2 expression, and increased expression of the remaining genes. Furthermore, inoculation with bacterial isolates improved the accumulation of Mn, Zn, and Ca in soybean tissues. In this study, B. licheniformis showed potential to be incorporated in formulations for improving soybean grown in calcareous soil. The formulation of BIs contemplate a series of requirements and their effective implementation is still challenging. However, they are a promising trend to the accomplishment of future global politics and present a series of advantages to greener agriculture practices that are critically reviewed in the last part of this thesis. In general, the results presented in this thesis contribute to better understand the mechanisms by which PGPB improve Fe uptake and plant growth, under alkaline conditions, and their potential as bioinoculants in a sustainable perspective.