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- 2-Fluorophenol degradation by aerobic granules in a sequencing batch reactorPublication . Duque, A. F.; Bessa, V. S.; Carvalho, M. F.; De Kreuk, M. K.; Loosdrecht, M. C. M. van; Castro, P. M. L.
- Performance of an aerobic granular sequencing batch reactor fed with wastewaters contaminated with Zn2+Publication . Marques, Ana P. G. C.; Duque, Anouk F.; Bessa, Vânia S.; Mesquita, Raquel B. R.; Rangel, António O. S. S.; Castro, Paula M. L.The main aim of this study was to investigate the performance of an aerobic granular sludge sequencing batch reactor (AGS-SBR) receiving water streams supplied with different loads of Zn2+ (50 and 100 mg L-1) during an operation of 866 cycles (ca. 109 days). When the metal was not fed, chemical oxygen demand (COD), PO43- and NH4+ were efficiently removed, with efficiencies of 56, 23 and 72% respectively. DGGE profiles showed that Zn2+ supply negatively affected the bacterial diversity and community structure of the granules. Consequently, the shock loadings with Zn2+, particularly at the higher levels (100 mg L-1), affected the nutrient removal in the AGS-SBR, although the reactor still generally complied with admissible legal values concerning organic matter, nitrogen and Zn. Simultaneous removal of PO43- and TSS in such conditions needs further refining but the application of aerobic granular SBR in the treatment of Zn2+ contaminated wastewaters seems viable.
- Isolation and characterization of a Rhodococcus strain able to degrade 2-fluorophenolPublication . Duque, Anouk F.; Hasan, Syed A.; Bessa, Vânia S.; Carvalho, Maria F.; Samin, Ghufrana; Janssen, Dick B.; Castro, Paula M. L.A pure bacterial culture able to utilize 2- fluorophenol (2-FP) as sole carbon and energy source was isolated by selective enrichment from sediments collected from a contaminated site in Northern Portugal. 16S rRNA gene analysis showed that the organism (strain FP1) belongs to the genus Rhodococcus. When grown aerobically on 2-FP, growth kinetics of strain FP1 followed the Luong model. An inhibitory effect of increasing 2-FP concentrations was observed with no growth occurring at 2- FP levels higher than ca. 4 mM. Rhodococcus strain FP1 was able to degrade a range of other organofluorine compounds, including 2-fluorobenzoate, 3-fluorobenzoate, 4-fluorobenzoate, 3-fluorophenol, 4-fluorophenol, 3-fluorocatechol, and 4-fluorocatechol, as well as chlorinated compounds such as 2-chlorophenol and 4-chlorophenol. Experiments with cell-free extracts and partially purified enzymes indicated that the first step of 2-fluorophenol metabolism was conversion to 3-fluorocatechol, suggesting an unusual pathway for fluoroaromatic metabolism. To our knowledge, this is the first time that utilization of 2-FP as a growth substrate by a pure bacterial culture is reported.
- Enrichment of bacterial strains for the biodegradation of diclofenac and carbamazepine from activated sludgePublication . Bessa, V. S.; Moreira, I. S.; Tiritan, M. E.; Castro, P. M. L.Carbamazepine and diclofenac have been pointed out as important markers for environmental pollution by pharmaceuticals. This study reports on the isolation of bacterial strains capable to degrade these micropollutants from activated sludge of a municipal wastewater treatment plant (WWTP). After selective enrichments, one strain able to degrade diclofenac and two strains able to degrade carbamazepine were isolated. The strains were identified by 16S rRNA gene sequencing. Strain Brevibacterium sp. D4 was able to biodegrade 35% of 10 mg L−1 of diclofenac as a sole carbon source; periodic feeding with acetate as a supplementary carbon source resulted in enhancing biodegradation to levels up to 90%, with a concomitant increase of the biodegradation rate. Strains Starkeya sp. C11 and Rhizobium sp. C12 were able to biodegrade 30% of 10 mg L−1 of carbamazepine as a sole carbon source; supplementation with acetate did not improve the biodegradation of carbamazepine by these strains. The activated sludge harboured bacteria capable to degrade the two top priority environmental contaminants and may be potentially useful for biotechnological applications.
- Carbamazepine is degraded by the bacterial strain Labrys portucalensis F11Publication . Bessa, Vânia S.; Moreira, Irina S.; Murgolo, Sapia; Mascolo, Giuseppe; Castro, Paula M. L.The occurrence of pharmaceuticals in the environment is a topic of concern. Carbamazepine (CBZ) is a widespread antiepileptic drug and due to its physical-chemical characteristics minimal removal is achieved in conventional water treatments, and thus has been suggested as a molecular marker of wastewater contamination in surface water and groundwater. The present study reports the biotransformation of CBZ by the bacterial strain Labrys portucalensis F11. When supplied as a sole carbon source, a 95.4% biotransformation of 42.69 μM CBZ was achieved in 30 days. In co-metabolism with acetate, complete biotransformation was attained at a faster rate. Following a target approach, the detection and identification of 14 intermediary metabolites was achieved through UPLC-QTOF/MS/MS. Biotransformation of CBZ by the bacterial strain is mostly based on oxidation, loss of -CHNO group and ketone formation reactions; a biotransformation pathway with two routes is proposed. The toxicity of untreated and treated CBZ solutions was assessed using Vibrio Fischeri and Lepidium sativum acute toxicity tests and Toxi-Chromo Test. The presence of CBZ and/or its degradations products in solution resulted in moderate toxic effect on Vibrio Fischeri, whereas the other organisms were not affected. To the best of our knowledge this is the first report that proposes the metabolic degradation pathway of CBZ by a single bacterial strain.
- Bioaugmentation of a rotating biological contactor for degradation of 2-fluorophenolPublication . Duque, Anouk F.; Bessa, Vânia S.; Carvalho, Maria F.; Castro, Paula M. L.The performance of a laboratory scale rotating biological contactor (RBC) towards shock loadings of 2-fluorophenol (2-FP) was investigated. During a period of ca. 2 months organic shock loadings of 25 mg L-1 of 2-FP were applied to the RBC. As no biodegradation of 2-FP was observed, bioaugmentation of the RBC with a 2-FP degrading strain was carried out and, along ca. 6 months, organic shock loadings within a range of 25-200 mg L-1 of 2-FP were applied. Complete biodegradation of 50 mg L-1 of 2-FP was observed during operation of the reactor. The RBC showed to be robust towards starvation periods, as after ca. 1 month of non-supply of the target compound, the reactor resumed 2-FP degradation. The inoculated strain was retained within the biofilm in the disks, as the 2-FP degrading strain was recovered from the biofilm by the end of the experiment, thus bioaugmentation was successfully achieved.
- Constructed wetland with a polyculture of ornamental plants for wastewater treatment at a rural tourism facilityPublication . Calheiros, Cristina S. C.; Bessa, Vânia S.; Mesquita, Raquel B. R.; Brix, Hans; Rangel, António O. S. S.; Castro, Paula M. L.Sewage management in remote rural and mountain areas constitutes a challenge because of the lack of adequate infrastructure and economic capability. Tourism facilities, in particular, possess a special challenge because of huge variability in sewage production and composition as a consequence of variations in number of guests and their activities. Constructed wetlands (CWs) are recognized as a robust and economical ecotechnology capable of meeting these challenges. A horizontal subsurface flow CW system was established at a guest house located in a rural and mountain area of Portugal. The substrate of the bed was an expanded clay substrate, and the system was planted with a polyculture of ornamental flowering plants (Canna flaccida, Zantedeschia aethiopica, Canna indica, Agapanthus africanus and Watsonia borbonica). The load and composition of sewage varied significantly seasonally (17–579 kg COD ha−1 d−1), but removal efficiencies of BOD and COD were generally high (>90%) and independent of the loading conditions. The system also reduced PO43− (up to 92%), NH4+ (up to 84%) and total coliform bacteria (up to 99%). The ornamental polyculture provided an aesthetic pleasing system with different appearance during the seasons. Of the five species tested, four grew well (C. flaccida, C. indica, Z. aethiopica and W. borbonica), whereas A. africanus was outcompeted. The system owner cut flowers from the CW system and used them for decorations at the guest house. It was demonstrated that CW systems planted with a polyculture of ornamental plant species, besides the water treatment function, possess several additional benefits including aesthetics and biodiversity enhancement.
- 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 dynamics in a rotating biological contactor treating 2‑fluorophenol‑containing wastewaterPublication . Duque, Anouk F.; Bessa, Vânia S.; Castro, Paula M. L.One of the main factors affecting the performance of rotating biological contactors (RBC) is the biofilm characteristics. Therefore, a deep understanding of the microbial population dynamics and structure of the biofilm is mandatory if optimization of organic matter and nutrients removal is targeted. This study focused on the effects of organic shock loads of 2-fluorophenol (2-FP) on the microbial diversity present in an RBC biofilm. The RBC was seeded with activated sludge from a conventional wastewater treatment plant and was operated during 496 days. During the first 126 days, the RBC was subjected to intermittent 2-FP shocks of 25 mg l(-1) and no degradation occurred. Therefore, the reactor was subsequently augmented with a 2-FP-degrading strain (FP1). Afterwards, the RBC had a stable performance when subjected to 2-FP shocks up to 50 mg l(-1) and to a starvation period, as indicated by removal of the compound. Denaturing gradient gel electrophoresis (DGGE) revealed large shifts in microbial communities present in the first and fifth stages, although no clear relation between the sample collection time and spatial factor was found. Phylogenetic affiliation of some predominant members was assessed by direct sequencing of correspondent DGGE bands. Affiliations to alpha-, beta- and delta-Proteobacteria were found. Several bacterial strains isolated from the reactor showed capacity for 2-FP degradation. Strain FP1 was successfully recovered from the biofilm by plating and by DGGE, reinforcing that bioaugmentation was successfully achieved.
- Biological removal processes in aerobic granular sludge exposed to diclofenacPublication . Bessa, Vânia S.; Moreira, Irina S.; Loosdrecht, Mark C. M. van; Castro, Paula M. L.Diclofenac is a worldwide consumed drug included in the watch list of substances to be monitored according to the European Union Water Framework Directive (Directive 2013/39/EU). Aerobic granular sludge sequencing batch reactors (AGS-SBR) are increasingly used for wastewater treatment but there is scant information on the fate and effect of micropollutants to nutrient removal processes. An AGS-SBR fed with synthetic wastewater containing diclofenac was bioaugmented with a diclofenac degrading bacterial strain and performance and microbial community dynamics was analysed. Chemical oxygen demand, phosphate and ammonia removal were not affected by the micropollutant at 0.03 mM (9.54 mg L-1). The AGS was able to retain the degrading strain, which was detected in the sludge throughout after augmentation. Nevertheless, besides some adsorption to the biomass, diclofenac was not degraded by the augmented sludge given the short operating cycles and even if batch degradation assays confirmed that the bioaugmented AGS was able to biodegrade the compound. The exposure to the pharmaceutical affected the microbial community of the sludge, separating the two first phases of reactor operation (acclimatization and granulation) from subsequent phases. The AGS was able to keep the bioaugmented strain and to maintain the main functions of nutrient removal even through the long exposure to the pharmaceutical, but combined strategies are needed to reduce the spread of micropollutants in the environment.