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- Long-term stability of a non-adapted aerobic granular sludge process treating fish canning wastewater associated to EPS producers in the core microbiomePublication . Paulo, Ana M. S.; Amorim, Catarina L.; Costa, Joana; Mesquita, Daniela P.; Ferreira, Eugénio C.; Castro, Paula M. L.The tolerance of aerobic granular sludge (AGS) to variable wastewater composition is perceived as one of its greatest advantages compared to other aerobic processes. However, research studies select optimal operational conditions for evaluating AGS performance, such as the use of pre-adapted biomass and the control of wastewater composition. In this study, non-adapted granular sludge was used to treat fish canning wastewater presenting highly variable organic, nutrient and salt levels over a period of ca. 8 months. Despite salt levels up to 14 g NaCl L−1, the organic loading rate (OLR) was found to be the main factor driving AGS performance. Throughout the first months of operation, the OLR was generally lower than 1.2 kg COD m−3 day−1, resulting in stable nitrification and low COD and phosphorous levels at the outlet. An increase in OLR up to 2.3 kg COD m−3 day−1 disturbed nitrification and COD and phosphate removal, but a decrease to average values between 1 and 1.6 kg COD m−3 day−1 led to resuming of those processes. Most of the bacteria present in the AGS core microbiome were associated to extracellular polymeric substances (EPS) production, such as Thauera and Paracoccus, which increased during the higher OLR period. Ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) species were detected in AGS biomass; while AOB were identified throughout the operation, NOB were no further identified after the period of increased OLR. Different polyphosphate-accumulating organisms (PAOs) were detected along the process: Candidatus Accumulibacter, Tetrasphaera and Gemmatimonas. A non-adapted granular sludge was able to treat the fish canning wastewater and to tolerate salinity fluctuations up to 14 g L−1. Overall, a high microbial diversity associated to EPS producers allowed to preserve bacterial groups responsible for nutrients removal, contributing to the adaptation and long-term stability of the AGS system.
- Simultaneous partial nitrification and 2-fluorophenol biodegradation with aerobic granular biomass: reactor performance and microbial communitiesPublication . Ramos, Carlos; Amorim, Catarina L.; Mesquita, Daniela P.; Ferreira, Eugénio C.; Carrera, Julián; Castro, Paula M. L.An aerobic granular bioreactor was operated for over 4months, treating a synthetic wastewater with a high ammonium content (100mgNL-1). The inoculum was collected from a bioreactor performing simultaneous partial nitrification and aromatic compounds biodegradation. From day-56 onwards, 2-fluorophenol (2-FP) (12.4mgL-1) was added to the feeding wastewater and the system was bioaugmented with a 2-FP degrading bacteria (Rhodococcus sp. FP1). By the end of operation, complete 2-FP biodegradation and partial nitrification were simultaneously achieved. Aerobic granules remained stable over time. During the 2-FP loading, a shift in the community structure occurred, coinciding with the improvement of 2-FP degradation. DGGE analysis did not allow to infer on the bioaugmented strain presence but pyrosequencing analysis detected Rhodococcus genus by the end of operation. Together with other potential phenolic-degraders within granules, these microorganisms were probably responsible for 2-FP degradation.
- Development of a low pressure chromatographic flow system for monitoring the biodegradation of ofloxacin and ciprofloxacinPublication . Santos, Inês C.; Mesquita, Raquel B. R.; Amorim, Catarina L.; Castro, Paula M. L.; Rangel, António O. S. S.In this work, we propose a simple low pressure chromatography method with a high throughput for monitoring the biodegradation of fluoroquinolones. Fluoroquinolones are a class of antibiotics that have been accumulating in the environment as a consequence of their release from different sources, namely hospital waste. It has been found that wastewater treatment plants are not able to completely remove this type of micro-pollutants and so, alternative solutions are necessary. Some biodegradation studies have been done but the HPLC-based methodologies used to monitor the degradation process usually require high cost instrumentation such as analytical columns and detectors. For this reason, a 1 cm monolithic column was coupled to a flow injection system and used for the simultaneous quantification of ofloxacin and ciprofloxacin with UV detection at 295 and 275 nm, respectively. LODs of 0.5 and 0.29 mg L-1 for ofloxacin and ciprofloxacin were obtained, respectively, with repeatability within the range of 2–10%. The developed method was successfully applied to monitor the biodegradation of ofloxacin and ciprofloxacin by the strain Labrys portucalensis F11. The results proved that the low pressure chromatography method is a simpler, cheaper, and faster alternative to monitor biodegradation studies.
- Biological treatment of a contaminated gaseous emission from a leather industry in a suspended-growth bioreactorPublication . Carvalho, M. F.; Duque, A. F.; Moura, S. C.; Amorim, Catarina L.; Ferreira Jorge, R. M.; Castro, Paula M. L.A suspended-growth bioreactor (SGB) was operated for the treatment of a gaseous stream mimicking emissions generated at a leather industrial company. The main volatile organic compounds (VOCs) present in the gaseous stream consisted of 1-methoxy-2-propanol, 2,6-dimethyl-4-heptanone, 2-butoxyethanol, toluene and butylacetate. A microbial consortium able to degrade these VOCs was successfully enriched. A laboratory- scale SGB was established and operated for 210-d with an 8 h cycle period and with shutdowns at weekends. Along this period, the SGB was exposed to organic loads (OL) between 6.5 and 2.3 £ 102 g h¡1 m¡3. Most of the compounds were not detected at the outlet of the SGB. The highest total VOC removal efficiency (RE) (ca 99%) was observed when an OL of 1.6 £ 102 g h¡1 m¡3 was fed to the SGB. The maximum total VOC elimination capacity (1.8 £ 102 g h¡1 m¡3) was achieved when the OL applied to the SGB was 2.3 £ 102 g h¡1 m¡3. For all the operating conditions, the SGB showed high levels of degradation of toluene and butylacetate (RE t 100%). This study also revealed that recirculation of the gaseous effluent improved the performance of the SGB. Overall, the SGB was shown to be robust, showing high performance after night and weekend shutdown periods.
- Variability in the composition of extracellular polymeric substances from a full-scale aerobic granular sludge reactor treating urban wastewaterPublication . Oliveira, Ana S.; Amorim, Catarina L.; Ramos, Miguel A.; Mesquita, Daniela P.; Inocêncio, Paulo; Ferreira, Eugénio C.; Loosdrecht, Mark van; Castro, Paula M. L.Within the framework of the circular economy, there is a need for waste management alternatives that promote the reuse of materials produced in wastewater treatment plants (WWTP). An interesting option is the recovery of extracellular substances from sludge. The variability of characteristics of potential recovered bioproducts has to be assessed in full scale operational settings. In this study, aerobic granular sludge (AGS) from a full-scale WWTP treating urban wastewater was regularly collected for 4 months to assess variability in extracellular polymeric substances (EPS) composition and in granular morphology. Variations in the EPS composition occurred with time. Proteins and humic substances were the main EPS components (329–494 and 259−316 mg/g VSS of AGS, respectively), with polysaccharides and DNA representing minor components. The application of an extra purification step after extraction to obtain a purer EPS led to a decrease in the yield of each EPS component, particularly pronounced for the polysaccharides. The final product had a rather constant composition for the monthly samples. The granules showed morphological stability throughout the sampling period and the yield of EPS was correlated to the size of the granules, higher when there was a higher content of small granules (Deq<150 μm) comparing to intermediate (150 ≤ Deq<1500 μm) or large granules (Deq≥1500 μm). This is the first time that a potential valorization strategy for surplus AGS biomass is studied in a full-scale environment. Knowledge on yield and product homogeneity is important as these features are essential for downstream application of the recovered EPS.
- Biodegradation of ofloxacin, norfloxacin, and ciprofloxacin as single and mixed substrates by Labrys portucalensis F11Publication . Amorim, Catarina L.; Moreira, Irina S.; Maia, Alexandra S.; Tiritan, Maria E.; Castro, Paula M. L.Fluoroquinolone (FQ) antibiotics are extensively used both in human and veterinary medicine, and their accumulation in the environment is causing an increasing concern. In this study, the biodegradation of the three most worldwide used FQs, namely ofloxacin, norfloxacin, and ciprofloxacin, by the fluoroorganic-degrading strain Labrys portucalensis F11 was assessed. Degradation occurred when the FQs were supplied individually or as mixture in the culture medium, in the presence of an easily degradable carbon source. Consumption of individual FQs was achieved at different extents depending on its initial concentration, ranging from 0.8 to 30 μM. For the lowest concentration, total uptake of each FQ was observed but stoichiometric fluoride release was not achieved. Intermediate compounds were detected and identified by LC-MS/MS with a quadrupole time of flight detector analyzer. Biotransformation of FQs by L. portucalensis mainly occurred through a cleavage of the piperazine ring and displacement of the fluorine substituent allowing the formation of intermediates with less antibacterial potency. FQ-degrading microorganisms could be useful for application in bioaugmentation processes towards more efficient removal of contaminants in wastewater treatment plants.
- Wastewater valorization by pure bacterial cultures to Extracellular Polymeric Substances (EPS) with high emulsifying potential and flocculation activitiesPublication . Drakou, Efi‑Maria; Amorim, Catarina L.; Castro, Paula M. L.; Panagiotou, Fostira; Vyrides, IoannisNowadays much effort has been devoted for the development of cost-effective and environmentally friendly processes to obtain extracellular polymeric substances (EPS) with high emulsifying and flocculation activities. The aim of this study was to evaluate the capacity of bacterial strains previously isolated from oil-contaminated areas to produce EPS with high emulsification and bioflocculant properties during cultivation in domestic and bilge wastewater and in industrial crude glycerol. A total of seven bacterial strains were screened for EPS production, from which two strains, Pseudomonas aeruginosa LVD-10 and Enterobacter sp. SW, were selected as potential EPS producers. EPS with high emulsifying capacity in olive oil (a maximum of 96.6 and 89.8% for strain SW and LVD-10, respectively) was produced using bilge wastewater as substrate. EPS with a slightly lower emulsifying capacity was obtained using crude glycerol. In addition, the flocculation activity of the EPS extracted from strains LVD-10 and SW grown on crude glycerol was considerably higher (81.6 and 73.3%, respectively) than that obtained with other substrates. This is the first study that points out that EPS with emulsifying and flocculation potential activity can be produced from bilge wastewater and crude glycerol. The production of biopolymers with broad biotechnological applications using low-cost substrates can be a means to valorise waste streams.
- MALDI-TOF MS for the identification of cultivable organic-degrading bacteria in contaminated groundwater near unconventional natural gas extraction sitesPublication . Santos, Inês C.; Martin, Misty S.; Carlton Jr., Doug D.; Amorim, Catarina L.; Castro, Paula M. L.; Hildenbrand, Zacariah L.; Schug, Kevin A.Groundwater quality and quantity is of extreme importance as it is a source of drinking water in the United States. One major concern has emerged due to the possible contamination of groundwater from unconventional oil and natural gas extraction activities. Recent studies have been performed to understand if these activities are causing groundwater contamination, particularly with respect to exogenous hydrocarbons and volatile organic compounds. The impact of contaminants on microbial ecology is an area to be explored as alternatives for water treatment are necessary. In this work, we identified cultivable organic-degrading bacteria in groundwater in close proximity to unconventional natural gas extraction. Pseudomonas stutzeri and Acinetobacter haemolyticus were identified using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), which proved to be a simple, fast, and reliable method. Additionally, the potential use of the identified bacteria in water and/or wastewater bioremediation was studied by determining the ability of these microorganisms to degrade toluene and chloroform. In fact, these bacteria can be potentially applied for in situ bioremediation of contaminated water and wastewater treatment, as they were able to degrade both compounds.
- Removal of fluoxetine and its effects in the performance of an aerobic granular sludge sequential batch reactorPublication . Moreira, Irina S.; Amorim, Catarina L.; Ribeiro, Ana R.; Mesquita, Raquel B. R.; Rangel, António O.S.S.; Loosdrecht, Mark C.M. van; Tiritan, Maria E.; Castro, Paula M. L.Fluoxetine (FLX) is a chiral fluorinated pharmaceutical mainly indicated for treatment of depression and is one of the most distributed drugs. There is a clear evidence of environmental contamination with this drug. Aerobic granular sludge sequencing batch reactors constitute a promising technology for wastewater treatment; however the removal of carbon and nutrients can be affected by micropollutants. In this study, the fate and effect of FLX on reactor performance and on microbial population were investigated. FLX adsorption/desorption to the aerobic granules was observed. FLX shock loads (<= 4 mu M) did not show a significant effect on the COD removal. Ammonium removal efficiency decreased in the beginning of first shock load, but after 20 days, ammonia oxidizing bacteria became adapted. The nitrite concentration in the effluent was practically null indicating that nitrite oxidizing bacteria was not inhibited, whereas, nitrate was accumulated in the effluent, indicating that denitrification was affected. Phosphate removal was affected at the beginning showing a gradual adaptation, and the effluent concentration was <0.04 mM after 70 days. A shift in microbial community occurred probably due to FIX exposure, which induced adaptation/restructuration of the microbial population. This contributed to the robustness of the reactor, which was able to adapt to the FLX load.
- Aerobic granular sludge has EPS-producing bacteria able to tolerate saltPublication . Paulo, Ana M. S.; Amorim, Catarina L.; Castro, Paula M. L.The aerobic granular sludge (AGS) process is a promising biotechnology which relies on the formation of compact biomass granules. Granulation occurs due to the overproduction of extracellular polymeric substances (EPS) by some microbes in response to stress conditions. EPS protect bacteria from the effect of toxic or inhibiting compounds present in the wastewater, such as salts. One of the current challenges is to use the AGS process to treat high salinity wastewater, commonly produced by agro-food and chemical industries. The main objective of this study was to screen for EPS-producing bacteria bacteria in an AGS reactor treating synthetic saline wastewater contaminated with a toxic compound. Several bacterial isolates were obtained from the reactor biomass. Genomic DNA was extracted and isolates (30) were grouped according to species similarity, based on RAPD profiles. Isolates displaying unique profiles (15) were subsequently identified by 16S rRNA gene sequencing analysis. Bacteria highly related to Pseudomonas, Aeromonas, Stenotrophomonas, Flavobacterium and Pseudoxanthomonas were obtained. Isolates SG4 (Stenotrophomonas) and FG10 (Flavobacterium) belong to bacterial genera associated to EPS production in granules. These were selected for growth and biofilm formation assays with increasing NaCl concentrations (0 to 35 g L-1). Both isolates were able to grow in the presence of 35 g NaCl L-1, despite at a lower growth rate. Although salt increase affected biofilm production, SG4 was the best biofilm producer. EPS production by SG4 in the presence of 10 and 20 g L-1 of NaCl was compared. EPS was extracted and the content in proteins, humic acids and carbohydrates was quantified. SG4 was able to produce more EPS in the presence of 10 g L-1 (123 mg g-1 VSS) compared to 20 g L-1 of NaCl (77.6 mg g-1 VSS). EPS-producing bacteria with ability to tolerate high salinity were retrieved from an AGS process treating synthetic wastewater. Further research is required to gain more knowledge on these bacteria and their importance for the robustness of a process treating saline wastewater.
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