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- Studies on enantioselective biodegradation of fluoxetinePublication . Moreira, Irina S.; Ribeiro, Ana R.; Amorim, Catarina L.; Tiritan, Maria E.; Castro, Paula M. L.Fluoxetine (FLX) is a chiral fluorinated pharmaceutical indicated mainly for treatment of depression and is one of the most dispensed drugs in the world. There is a clear evidence of environmental contamination with this drug. Granular sludge sequencing batch reactors (SBR) constitute a promising technology for the treatment of effluents containing micropollutants. In this study, a SBR was operated in order to assess its performance when treating a synthetic wastewater containing racemic FLX (rac-FLX), under continuous and intermittent feeding of the compound. The concentration of FLX enantiomers was followed using an enantioselective HPLC method. A removal of 70% of the total supplied FLX was observed in the first continuous feeding period. However, the subsequent feeding periods revealed a significant decrease in the FLX removal; FLX liberation occurred during periods when no compound was supplied. This can be probably explained by desorption of FLX previously adsorbed to the granules. No intermediate metabolites or fluoride release were detected, corroborating the hypothesis that adsorption of FLX to the aerobic granules occurred. Moreover, the absence of enantioselectivity in the decrease of FLX enantiomers concentration is also an indicator of an abiotic mechanism. In face of the incapacity of the aerobic granules to biodegrade FLX, the ability of Labrys portucalensis F11, a previously isolated microbial strain with the capacity to degrade a range of fluorinated aromatic compounds, to biodegrade this compound was investigated. In this study, the enantioselective biodegradation of rac-FLX and of its enantiomers was assessed. The results obtained revealed that this strain is able to degrade both enantiomers of FLX, when supplemented as a racemic mixture, as well as when supplemented as a single enantiomers. Preferential degradation of the (R)-enantiomer was observed. This feature makes L. portucalensis F11 a potential candidate for devising biodegradation technologies able to deal with contamination by this pharmaceutical. Acknowledgements: I.S. Moreira, A.R. Ribeiro and C.L. Amorim wish to acknowledge a research grant from Fundação para a Ciência e Tecnologia (FCT), Portugal (Ref. SFRH/BPD/87251/2012, SFRH/BD/64999/2009 and SFRH/BD/47109/2008 , respectively) and Fundo Social Europeu (Programa Operacional Potencial Humano (POPH), Quadro de Referência Estratégico Nacional (QREN)). This work was supported by FCT through the projects PTDC/EBB-EBI/111699/2009, CEQUIMED-Pest-OE/SAU/UI4040/2011 and PEst-OE/EQB/LA0016/2011.
- Aerobic granular sludge process treats real fish canning wastewater.Publication . Paulo, A. M. S.; Amorim, Catarina L.; Castro, P. M. L.Aerobic Granular Sludge (AGS) is an innovative technology used for carbon and nutrients removal from wastewater, using less space and energy compared to other biotechnological solutions. Aerobic granules present a compact structure, composed of extracellular polymeric substances (EPS), which increase AGS resistance to variable wastewater composition, as those commonly produced by industry. In this study, C, N and P-removal from a fish canning plant wastewater was evaluated using an AGS-SBR (sequential batch reactor). Throughout the first 3 months of operation with variable feed composition, the chemical oxygen demand (COD) at the outlet was below the discharge limit of 125 mg O2 L-1, phosphorous removal was stable and the nitrification process improved. At the higher organic loading rates (OLR), the AGS performance was temporary affected. This study contributes to understanding the effects of the variability of a real wastewater on an AGS process.
- Bacterial community dynamics within an aerobic granular sludge reactor treating wastewater loaded with pharmaceuticalsPublication . Amorim, Catarina L.; Alves, Marta; Castro, Paula M.L.; Henriques, IsabelPharmaceuticals are micropollutants often present in wastewater treatment systems. In this study, the potential impact of such micropollutants on the bacterial population within aerobic granular sludge (AGS) bioreactor was investigated. The AGS bacterial community structure and composition were accessed combining DGGE fingerprinting and barcoded pyrosequencing analysis. Both revealed the existence of a dynamic bacterial community, independently of the pharmaceuticals presence. The AGS microbiome at both phylum and class levels varied over time and, after stopping pharmaceuticals feeding, the bacterial community did not return to its initial composition. Nevertheless, most of the assigned OTUs were present throughout the different operational phases. This core microbiome, represented by over 72% of the total sequences in each phase, probably played an important role in biological removal processes, avoiding their failure during the disturbance period. Quantitative-PCR revealed that pharmaceuticals load led to gradual changes on the abundance of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and polyphosphate-accumulating organisms (PAO) but their persistence during that phase demonstrated the resilience of such bacterial groups. AGS microbiome changed over time but a core community was maintained, probably ensuring the accomplishment of the main biological removal processes.
- Specialized degrading granules effective for biaugmentation of Aerobic Granular Sludge reactor treating 2-fluorophenol in wastewaterPublication . Oliveira, Ana S.; Amorim, Catarina L.; Zlopasa, Jure; Lin, Yumei; Loosdrecht, Mark C. M. van; Castro, PaulaThe amount of industrial chemicals being released into the environment has increased. Indigenous microbial communities in wastewater biotreatment processes are not always effective in removing xenobiotics. This work aimed to evaluate the feasibility and efficiency of a promising bioaugmentation strategy in an aerobic granular sludge (AGS) system continuously fed with 2-fluorophenol (2-FP). Bioreactor performance in terms of phosphate and ammonium removal and 2-FP degradation was evaluated. Granules were produced using extracellular polymeric substances (EPS) extracted from AGS as a carrying matrix and a 2-FP degrading strain, Rhodococcus sp. FP1. Afterwards, the produced granules were introduced in the reactor. Shortly after addition, the produced granules broke down into smaller fragments inside the bioreactor, but 2-FP degradation occurred. After 8 days of bioaugmentation, 2-FP concentration inside the reactor started to decrease, and stoichiometric fluorine release was observed 35 days later. 14 Days after the bioaugmentation, phosphate and ammonium removal efficiency improved ca. 36% and 48%, respectively. However, complete phosphorous and ammonium removal was never achieved while the reactor was fed with 2-FP. The persistency of Rhodococcus sp. FP1 in the reactor was followed by qPCR. Rhodococcus sp. FP1 was detected 1 day after in the AGS and up to 3 days after bioaugmentation at the effluent. Nevertheless, the degradative ability remained thereafter in the granules. Degrading strain could have persisted even if at lower numbers. Horizontal gene transfer could have happened from the 2-FP degrading strain to indigenous microbiome as some bacteria isolated from the AGS, 3 months after bioaugmentation, degraded 2-FP.
- Strategies to enhance the removal of FluoroquinolonesPublication . Amorim, Catarina L.; Maia, Alexandra S.; Moreira, Irina S.; van Loosdrecht, Mark C.M.; Tiritan, Maria E.; Castro, Paula M.L.Fluoroquinolones (FQs) are broad-spectrum antibiotics that play an important role in the treatment of serious bacterial infections. Currently, several FQs are available but ciprofloxacin (CPF), ofloxacin (OFL) and norfloxacin (NOR) are amongst the most worldwide prescribed antibiotics. Antibiotics can reach wastewater treatment plants (WWTP) from different routes. Thus removal of these contaminants during the biotreatment process is of major importance in order to avoid their release to other environmental matrices. Granular sludge sequencing batch reactors (SBR) constitute a novel biofilm technology for wastewater treatment extremely promising for the treatment of effluents containing toxic compounds. Therefore, in this study a granular sludge SBR, established with activated sludge from a WWTP, was operated for the treatment of an aqueous stream containing FQs. No evidence of FQ biodegradation followed by HPLC with Fluorescence Detection was observed but FQs adsorbed to the aerobic granular sludge, being gradually released into the medium after withdrawal of the FQs in the inlet stream. In a previous study, Labrys portucalensis F11 demonstrated to be able to degrade FQs, namely OFL, NOR and CPF, when supplied individually or as a mixture, in the presence of an easy degradable carbon source. Different removal extents were obtained for the tested concentrations (ranging from 0.8 to 30 μM), but overall the uptake capacity of strain F11 for individual FQs decreased with increasing the initial FQ concentration. When supplied with a mixture FQs, strain F11 concomitantly removed each target antibiotic but a decrease on the biodegradability of FQs was observed which could be explained by competition mechanisms. The ability of Labrys portucalensis F11 to grow using the readily available carbon source while maintain its ability to degrade FQs reinforce the potential of this strain in bioaugmentation processes. As the indigenous microbial communities in biotreatment processes rarely are able to remove such contaminants, using this promising FQ-degrading strain, bioaugmentation strategies such as inoculation of the degrading strain, as a suspension or immobilized on carrier material, or using a plasmid donor strain carrying the degradative genes, could be assessed to improve FQ removal. Acknowledgments: C.L. Amorim, A.S. Maia and I.S. Moreira wish to acknowledge the research grants from Fundação para a Ciência e Tecnologia (FCT), Portugal (Ref. SFRH/BD/47109/2008, SFRH/BD/86939/2012 and SFRH/BPD/87251/2012, respectively) and Fundo Social Europeu (Programa Operacional Potencial Humano (POPH), Quadro de Referência Estratégico Nacional (QREN))). This work was supported by FCT through the projects PTDC/EBB-EBI/111699/2009 and PEst-OE/EQB/LA0016/2011.
- Fluoroquinolones biosorption onto microbial biomass: activated sludge and aerobic granular sludgePublication . Ferreira, Vanessa R.A.; Amorim, Catarina L.; Cravo, Sara M.; Tiritan, M. E.; Castro, P. M. L.; Afonso, Carlos M. M.Fluoroquinolones (FQs) have been reported in trace amounts in different environmental matrices. The biosorption of three most prescribed FQs, ofloxacin (OFL), norfloxacin (NOR) and ciprofloxacin (CPF) by activated sludge (AS) and aerobic granular sludge (AGS) was investigated. Biosorption assays were conducted with FQs concentrations within the range of 100e700 ng mL 1, to mimic environmental conditions. At neutral pH and at the end of 48 h, AS showed higher biosorption capacity than AGS. For AS, a maximum biosorption of 1.50 ± 0.03, 3.24 ± 0.05 and 3.39 ± 0.06 mg gTSS 1 was observed for OFL, NOR and CPF respectively, whereas for AGS the maximum amount of FQs biosorbed was 1.18 ± 0.03, 2.73 ± 0.02 and 2.94 ± 0.03 mg gTSS 1. Langmuir isotherm was more applicable for describing FQs biosorption equilibrium by AS while for AGS, the Freundlich isotherm was more adequate. Given the AGS technology innovative character, the effect of change of pH on the biosorbed FQs was evaluated. FQs could be desorbed from AGS at pH 3, pH 8 and pH 9 but at pH 4 the biosorption process was promoted. This study allows a better understanding of the FQs biosorption processes. Moreover, the data from biosorption/desorption from AGS may be useful for management and operation of AGS bioreactors
- Bioaugmentation of Aerobic Granular Sludge with specialized degrading granules treating 2-fluorophenol wastewaterPublication . Oliveira, Ana S.; Amorim, Catarina L.; Zlopasa, Jure; Lin, Yumei; Loosdrecht, Mark C. M. van; Castro, PaulaThe industry growth has been accompanied by an increase in the amount of industrial chemicals being released into the environment. Indigenous microbial communities in wastewater biotreatment processes are not always effective in removing xenobiotics. This work aimed to evaluate the efficiency of a new bioaugmentation strategy in an aerobic granular sludge sequencing batch reactor (AGS-SBR) system fed with 2-fluorophenol (2-FP). Bioreactor performance in terms of phosphate and ammonium removal, 2-FP degradation and chemical oxygen demand (COD) was evaluated. The new bioaugmentation strategy consisted in producing granules using extracellular polymeric substances (EPS) extracted from AGS as a carrying matrix and a 2-FP degrading strain, Rhodococcus sp. FP1. The produced granules were used for the bioaugmentation of a reactor fed with 2-FP. Shortly after bioaugmentation, the produced granules broke down into smaller fragments inside the bioreactor, but 2-FP degradation occurred. After 8 days of bioaugmentation, 2-FP concentration inside the reactor started to decrease, and stoichiometric fluorine release was observed 35 days later. Phosphate and ammonium removal also improved after bioaugmentation, increasing from 30% to 38% and from 20 to 27%, respectively. Complete ammonium removal was only achieved when 2-FP feeding stopped, and phosphate removal was not recovered during operation time. COD removal also improved after the addition of the produced granules. The persistence of Rhodococcus sp. FP1 in the reactor was followed by qPCR. Rhodococcus sp. FP1 was detected 1 day after in the AGS and up to 3 days after bioaugmentation at the effluent. Nevertheless, the 2-FP degradative ability remained thereafter in the granules. Horizontal gene transfer could have happened from the 2-FP degrading strain to indigenous microbiome as some bacteria isolated from the AGS, 3 months after bioaugmentation, were able to degrade 2-FP. This study presents a promising and feasible bioaugmentation strategy to introduce specialized bacteria into AGS systems treating recalcitrant pollutants in wastewater.
- Characterization of the bacterial communities of aerobic granules in a 2-fluorophenol degrading processPublication . Duque, Anouk F.; Bessa, Vânia S.; Castro, Paula M. L.Aerobic granular sludge constitutes a novel technology for wastewater treatment. This study focused on the effect of 2-fluorophenol (2-FP) shock loadings on the microbial community diversity present in aerobic granules before and after inoculation with a bacterial strain able to degrade 2-FP, Rhodococcus sp. strain FP1. After bioaugmentation, apart from strain FP1, five culturable bacteria were isolated from the 2-FP degrading granules, belonging to the following genera: Serratia, Chryseobacterium, Xanthomonas, Pimelobacter and Rhodococcus. The latter two isolates are able to degrade 2-FP. Changes in the aerobic granules’ bacterial communities related to 2-FP shock loadings were examined using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pool. Numerical analysis of the DGGE profiles showed high diversity with an even distribution of species. Based on cluster analysis of the DGGE profiles, the bacterial communities present in the aerobic granules changes were related to the sampling time and the 2-FP concentration fed.
- Bacterial community and system performance of an aerobic granular sludge reactor treating pharmaceutical wastewaterPublication . Amorim, Catarina L.; Moreira, Irina S.; Ribeiro, Ana R.; Tiritan, Maria E.; Henriques, Isabel S.; Castro, Paula M.L.Pharmaceuticals often reach wastewater treatment systems where low removal rates are observed. In the present study the potential impact of a mixture of such micro-pollutants on an aerobic granular sludge-sequencing batch reactor (AGS-SBR) was investigated using a lab-scale bioreactor. COD and P- removals were affected due to the load of pharmaceuticals resulting in a decrease of the COD uptake and the P-release during the anaerobic feeding phase, but the discharge limits were not exceeded. Nevertheless, both processes returned to its normal operation after resuming the pharmaceuticals feeding. The nitrification process was also affected but the activity of bacteria responsible for both nitrification steps was able to recover. The exposure to the pharmaceuticals induced alterations in the bacterial community structure.
- Bacterial diversity shifts in AGS reactor treating food industry wastewaterPublication . Paulo, Ana M. S.; Amorim, Catarina L.; Castro, Paula M. L.Aerobic granular sludge (AGS) is a promising technology for treating industrial wastewater, possessing higher biomass retention and tolerance to toxic substrates than conventional activated sludge systems. AGS presents a diverse microbial community responsible for the simultaneous removal of carbon and nutrients. These communities are protected by extracellular polymeric substances (EPS) that allow for the compact structure of the granules. As a result, bacteria present in the aerobic granules are more resistant to variable wastewater composition, as commonly produced in food industry. The main objective of this work is to study the microbial community dynamics of an AGS reactor treating wastewater from a fish canning plant. The reactor was monitored during 220 days, divided into eight operational phases. COD, NH4+ and PO43- removal were assessed and biomass samples were collected throughout time for microbiome profiling. The reactor presented good COD, PO43- and NH4+ removal during phases I, II and III, but decreased performance during phase IV, when a higher organic load was applied. The removal processes recovered after phase IV until the end of operation. Proteobacteria were dominant in the inoculum (relative abundance of 64.8 %) and dominated almost all reactor phases. Bacteroidetes were second dominant in the inoculum (17.5 %) as well in most reactor phases, being present with higher relative abundance (55.5 %) than Proteobacteria (38.4 %) during phase IV. Within Proteobacteria, Gammaproteobacteria were initially more abundant but Betaproteobacteria predominated after phase IV. For Bacteroidetes, the community dynamics has also changed from phase IV onwards, with Flavobacteriia losing its high relative abundance to Saprospiria and Cytophagia. Several bacterial genera were detected throughout reactor operation, such as Phenylobacterium and Flavobacterium, while other were detected with higher abundance before (Methylocaldum and Plasticicumulans) or after phase IV (Thauera and Paracoccus). The relationship between bacterial community shifts and process performance was assessed. This study increases our knowledge on AGS technology application in real wastewater treatment.