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- Advanced treatment in constructed wetlands: the role of photocatalytic modules and nanostructured filtersPublication . Ojediran, Adetunji A.; Pereira, Sofia I. A.; Rosa-Santos, Paulo; Arenas, Francisco; Dolbeth, Marina; Ntougias, Spyros; Calheiros, Cristina S. C.Background & Aim: Constructed wetlands (CWs) have emerged as a leading nature-based solution (NBS) for wastewater treatment, offering multiple benefits such as low maintenance costs, high pollutant removal efficiency, carbon sequestration, biodiversity enhancement, and aesthetic value (Justino et al., 2023; Kadlec, 2008). However, in tourism facilities, two major challenges affect wastewater management: seasonal variability in wastewater composition and volume, and the lack of connection to centralized treatment systems (Calheiros et al., 2015). To address these issues, a decentralized CW system was developed, operating alongside a septic tank for wastewater collection. This horizontal subsurface flow CW has been operational since 2010. The study aims to integrate photocatalytic modules and nanostructured filter technologies with CW to enhance water quality for irrigation. Methods: wastewater quality characterization will be performed according to national legislation and the efficiency of the photocatalytic modules and nano filters will be assessed. Results: CW efficiency is being assessed, and ongoing studies are being carried out related to the best approach for the implementation of the photocatalytic modules and nanostructured filters technologies. Conclusions: By integrating advanced filtration and photocatalytic technologies, this research intends to develop a highly efficient, self-sustaining, and ecofriendly wastewater treatment system suitable for the tourism industry. The findings will contribute to the broader application of CW in decentralized wastewater management and sustainable water reuse strategies, most especially in a tourism facility.
- Aerobic granules synthesized with EPS and degrading strain Rhodococcus sp. FP1 for industrial wastewater treatmentPublication . Oliveira, Ana; Amorim, Catarina L.; Zlopassa, Jure; Lin, Yuemei; van Loosdrecht, Mark; Castro, Paula M. L.Background: Aerobic granular sludge-sequencing batch reactors (AGS-SBR) have been introduced as a promising and innovative wastewater treatment system economically outcompeting the conventional activated sludge system. AGS is considered a special case of suspended biofilms, composed of self-immobilized microorganisms that form spherical sludge aggregates. Microorganisms are embedded in a self-produced extracellular polymeric substances matrix thus avoiding the need of any carrier. Many synthetic organic chemicals or metabolites are being released directly into the environment after wastewater treatment processes, which are not conceived to remove them from the effluents. In spite of the AGS tolerance to toxicity, the indigenous microbial communities in biotreatment processes may not be effective in removing recalcitrant pollutants. Bioaugmentation strategies, which consists of adding specific microorganisms to the system can be a solution to overcome the difficulty to eliminate certain compounds in wastewaters. However, it is still not a well-established strategy and deserves attention to become a promising alternative. This work aimed to synthesize strong aerobic granules using EPS (extracted from AGS) and a specialized bacterial strain capable of degrading 2-fluorophenol, with the aid of specific substances to increase the cross-linking potential of EPS. Conclusions: Substance 1 was not efficient in improving the strength of the granules, due to concentration or inability to promote the crosslinking of EPS; Granules from mixture 6 were the most similar in terms of strength to the Utrecht granules. The source of EPS affected the strength of the granules, even with conditions similar to mixture 6 Control beads from mixture 7 showed to be weaker than the ones from mixture 6, reinforcing that EPS was a key component to increase the strength of the produced granules. EPS composition and concentration are important to produce strong granules that can be further used in bioaugmentation.
- Antimicrobial resistance in urban wastewater: insights from operating conditions and bacterial community dynamicsPublication . Ribeiro, Diana; Ruivo, Afonso; Santos, Isabel; Mena, Cristina; Vaz-Moreira, Ivone; Costa, Cláudio; Faria, Gabriela; Campinas, Margarida; Mesquita, Elsa; Silva, Catarina; Rosa, Maria João; Manaia, Célia M.
- Assessment of trombidiidae (acari) as biondicators for wasterwater treatment in a constructed wetlandPublication . Magalhães, João Pedro Correia de Sousa; Pereira, Sofia I. A.; Leong, Chi Man; Xu, John Hongxi; Calheiros, CristinaThe family Trombidiidae (subclass Acari), commonly known as red velvet mites, exhibits dietary habit shifts throughout their development. Larvae are parasitic, while nymphs and adults transition to a free - living, soil - dwelling predator stage. Predation by these mites is usually done in rocks, tree stumps, plants, leaf litter, and moss, with other arthropods and their eggs being their prey. These hunting environments fit the habitat created by constructed wetlands (CWs) biological wastewater treatment systems, mimicking the processes and conditions that occur in natural wetlands. Trombidiidae are recognized for their potential as bioindicators due to their sensitivity to a range of environmental factors. The presence of Trombidiidae was confirmed in all seasons in a 15-year - old CW located at a rural tourism house, implying that this CW maintains favorable environmental conditions year round. The simultaneous occurrence of spiders within the same system indicates that their life cycle is likely sustained within this system. As both parasites and predators of the biodiversity presented in the CW, Trombidiidae may contribute to a deeper understanding of the food web within these systems, and provide proof as bioindicators of the ecological and habitat benefits CWs can provide. This work involved seasonal sampling of macrofauna at multiple collection spots within and around the CW, complemented by substrate core sampling to assess belowground communities. The collected specimens were sorted, identified, and quantified, with statistical analysis currently underway. Preliminary results indicate a robust and well - structured ecosystem, with the consistent presence of Trombidiidae across all seasons, suggesting a stable population.
- Assessment of trombidiidae (acari) as biondicators for wastewater treatment in a constructed wetlandPublication . Magalhães, João Pedro Correia de Sousa; Pereira, Sofia; Leong, Chi Man; Xu, John Hongxi; Calheiros, Cristina S. C.Introduction and aim: The family Trombidiidae (superorder Acariformes), with common names such as red velvet mites, exhibits dietary habit shifts throughout their development. Larvae are parasitic, while nymphs and adults transition to a free-living, soil-dwelling predator stage (Durkin et al., 2021). Predation by these mites is usually done in rocks, tree stumps, plants, leaf litter, and moss, with other arthropods and their eggs being their prey. These hunting environments fit the habitat created by constructed wetlands (CWs) biological wastewater treatment systems, mimicking the processes and conditions that occur in natural wetlands. A macrofauna assessment was performed in a CW located in a tourism house in the north of Portugal, near Ponte de Lima, to understand its performance after 15 years of operation, with Trombidiidae mites being found. Method: This work involved seasonal sampling of macrofauna at multiple collection spots within and around the CW, with pitfall trapping being complemented by substrate core sampling to assess both belowground and aboveground communities. This occurred across the Autumn of 2022 to the Summer of 2023. The collected specimens were sorted by taxonomic group, identified further, when possible, photographed and quantified, with statistical analysis currently underway. Results and discussion: The presence of Trombidiidae was confirmed in all seasons in the 15-year-old CW, implying that this CW maintains favorable environmental conditions year-round. This tracks with the hypothesis of the environment created by the CW mimicking the natural habitats in which these predators hunt. The simultaneous occurrence of spiders, preferred hosts for the larval stage of these mites, within the same system indicates that their life cycle is likely sustained within this system. The fact that these spiders are numerous might also imply a robustness in their population, with their numbers not being heavily affected by the parasitism. As both parasites and predators of the biodiversity presented in the CW, Trombidiidae may contribute to a deeper understanding of the food web within these systems, providing proof as bioindicators of the ecological and habitat benefits CWs can provide. Conclusion: Preliminary results indicate a robust and well-structured ecosystem, with the consistent presence of Trombidiidae across all seasons, suggesting a stable population, with hosts for the larval stage, ensuring their cycle of life.
- 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.Pharmaceutical compounds are emerging environmental contaminants that have been detected in various environmental matrices [1]. Their occurrence has been widely reported in domestic and hospital wastewater treatment plants (WWTP) effluents where low removal rates are observed. Even at low levels, they can affect the composition of the microbial communities, and hence disturb the metabolic networks [2]. Aerobic granular sludge (AGS) has been successfully applied for the treatment of industrial and domestic wastewater and the implementation off AGS full-scale facilities has been growing worldwide [3]. The possibility to concomitantly remove carbon, nitrogen and phosphorous in a single unit is one of the attractive aspects of the AGS technology since it greatly reduces the plant footprint. Pharmaceuticals are often present in influent wastewaters, therefore knowledge on their effect on the main biological processes is needed to provide guidance for a stable operation. Moreover, as microorganisms within AGS are key players in removal processes, a better understanding of bacterial composition could be useful to improve wastewater treatment efficiency and to enlarge the knowledge on critical functional groups.
- Bacterial population variability in wastewater treatment and the potential persistence of specific lineagesPublication . Manaia, Célia; Ribeiro, Diana; Ruivo, Afonso; Santos, Isabel; Mena, Cristina; Vaz-Moreira, Ivone; Rosa, Maria João; Campinas, Margarida; Mesquita, Elsa; Silva, Catarina; Costa, Cláudio; Faria, GabrielaWastewater treatment plants (WWTPs) are essential barriers to attenuate the emission of contaminants, such as antibiotic-resistant bacteria (ARB) and resistance genes (ARGs), which monitoring is becoming a requirement (Directive (EU) 2024/3019). Despite the urgent need to improve wastewater treatment to remove ARB&ARGs, the complexity of microbial community dynamics during treatment remains poorly understood. This work discusses the bacterial community variations in full-scale real-world systems: i) between inlet and outlet of a WWTP with three full-scale activated sludge treatment lines, ii) due to ozonation, and iii) impact on the receiving river. Analyses included total coliforms and E. coli counts, quantitative PCR of selected genes (e.g. 16S rRNA, IS26, blaOXA-58, blaVEB), 16S rRNA gene metabarcoding, isolates identification by Maldi-TOF, and genome-based inference of ARB dissemination. Activated sludge treatment led to important variations on bacterial community composition, being genera affiliated to the phylum Candidatus Saccharibacteria, followed by those affiliated to the class Betaproteobacteria those that most frequently increased after treatment. Members of the genera Acinetobacter, Aeromonas and Aliarcobacter were among the most abundant in the inlet and persisted among the most abundant in the outlet. Other genera such as Bacteroides, Enterococcus, Parabacteroides, Streptococcus or Trichococcus decreased with the treatment. Ozonation was associated with the increase of members of the genera Acidovorax, Flavobacterium and Romboutsia and post-storage reactivation with the increase of Cloacibacterium and Flavobacterium and decrease of Romboutsia. Considering a group of 12 ARGs and MGEs, abundance decrease was observed after the activated sludge treatment and no impact was observed in the receptor river. The abundance (copies/mL) of the genes sul1 and blaOXA-58 increased after storage of the ozonated effluent up to 0.9 log-units. Presumptive coliforms isolated (~400) on CCA (Chromogenic Coliform Agar) or on CCA supplemented with cefotaxime (4 mg/L) were identified as Escherichia coli, Klebsiella pneumoniae, Citrobacter spp., and other and 16.4% (72/439) were multidrug resistant. The whole genome sequence of Klebsiella pneumoniae and Citrobacter freundii recovered from the inlet, outlet, after ozonation and river suggest clonal dissemination, for example a K. pneumoniae ST29 with plasmids ColRNAI and incFIB(K) from the WWTP to the river.
- Constructed wetland: multi-functional benefits of an ecological engineered systemPublication . Ojediran, Adetunji; Pereira, Sofia Almeida; Rosa-Santos, Paulo; Rodrigues, Ana Cristina; Calheiros, CristinaIntroduction: Constructed wetlands (CWs) are engineered systems that are inspired by natural wetlands [1]. They are often applied for decentralized wastewater treatment, but while their performance in pollutant removal is well established, other potential ecosystem services are often underappreciated. This study aims to bring attention to the broader multifunctional benefits of CWs —beyond treatment —by exploring a real - scale horizontal subsurface flow CW (HSSF - CW) located at a tourism facility in northern Portugal. Method: The CW, in operation since 2010, serves a rural guesthouse in Calheiros and consistently treats domestic wastewater generated from the guesthouse’s occupancy of between 6 and 40 people. The system covers an area of 40.5 m² and is vegetated with a polyculture of ornamental species, which is primarily for wastewater treatment at the facility but has also served aesthetic purposes. The CW operates under Mediterranean climatic conditions and discharges into a small polishing pond. Previous studies at this site have focused on pathogen removal and microbial communities [2], [3]. The current investigation builds on this foundation by identifying and evaluating other potential ecosystem functions. Results: The system’s stable operation provides a unique opportunity to assess additional services such as carbon sequestration (using biomass and substrate), energy recovery (from microbial fuel cell), soil amendments (biomass composting), safe water reuse for irrigation, and possibility of enhanced wastewater treatment (using photocatalytic modules and nanostructured filters). These aspects are now being explored to better understand the full value of CWs in multifunctional, real -world contexts. Conclusion: Recognizing the broader ecosystem functions of CWs is essential to reposition them as more than just wastewater treatment technologies. This study uses a successful, long - running HSSF - CW as a platform to investigate underexplored multifunctional benefits to show CWs's role as valuable ecosystem infrastructure —especially in decentralized settings like tourism where aesthetics, sustainability, and recovery of resources are essential.
- Constructed wetland: multi-functional benefits of an ecological engineered systemPublication . Ojediran, Adetunji; Pereira, Sofia; Rosa-Santos, Paulo; Rodrigues, Ana; Calheiros, Cristina S. C.Introduction and aim: Constructed wetlands (CWs) are engineered systems inspired by natural wetlands and commonly used for decentralized wastewater treatment. Although their efficiency in pollutant removal is well recognized, other ecosystem services they provide are often overlooked. This work highlights these broader multifunctional benefits through a real-scale horizontal subsurface flow CW (HSSF-CW) operating at a tourism facility in northern Portugal. Results and discussion: System has shown stable long-term operation, providing a reliable basis to explore additional ecosystem services beyond conventional wastewater treatment. Carbon sequestration is being assessed through biomass accumulation and carbon retention in the substrate. Energy recovery is under evaluation using microbial fuel cell integration Harvested plant biomass is being tested for composting and soil amendment, supporting nutrient recycling within the system. The treated effluent demonstrates potential for safe water reuse in irrigation, promoting circular water use on site. Treatment performance is being enhanced using photocatalytic and nanostructured filter modules. Future research would be advanced system optimization and modeling. Conclusion: Recognizing other broader functions of CWs is essential to repositioning them as more than just wastewater treatment technology but rather as multifunctional ecosystem systems that they are.
- Dynamics and interrelationships between antibiotic resistance, organic micropollutants and bacterial communities in full-scale rural constructed wetlandsPublication . Teixeira, A. Margarida; Matos, Diana; Coelho, Norberta; Halwatura, Lahiruni M.; Vaz-Moreira, Ivone; Castro, Paula M. L.; Aga, Diana S.; Manaia, Célia M.Constructed wetlands systems (CWs) are increasingly regarded as promising alternatives or complements to conventional wastewater treatment processes. However, the fate of chemical and biological contaminants in realworld treatment processes is understudied in this type of systems. This study aimed to fill this gap by evaluating the response of three horizontal subsurface flow CWs, in Northern Portugal, planted with Phragmites australis, in operation for >7 years, to reducing the load of fecal contamination, antibiotic resistance genes and organic micropollutants (OMPs). Influent, effluent and sediments samples (n = 36) were examined for abundance of cultivable Escherichia coli and total coliforms, total bacteria (16S rRNA gene), 10 genetic biomarkers associated with anthropogenic contamination (uidA, crAssphage, intI1, sul1, ermB, ermF, mefC, qacEΔ1, tetX and aph(3″)-Ib) by quantitative PCR, non-target LC-MS of OMPs and 16S rRNA gene-based bacterial community analysis. The three CWs showed reduction values (log-units/mL) up to 4.8 of E. coli and 3.6 of biomarkers, with the highest values observed in warmer periods. No evidence of for the accumulation microbiological contaminants in the sediments was observed. Among the 59 OMPs detected, reduction rates varied, and the concentration of the most abundant pharmaceutical compounds in the final effluent varied –reaching ng/L concentrations of ~36 000 for fenofibric acid, ~14 000 for acetaminophen, ~3000 for oxazepam and ~2000 for irbesartan, which can be considered high to discharge in the receiving environment. The bacterial community was dominated by members of the class Gammaproteobacteria, with treatment contributing to significant reduction of the relative abundance of members of the classes Clostridia, Bacilli and Actinomycetes. Compared with wastewater, sediments had significantly higher relative abundance of Alphaproteobacteria. The study confirms that CWs are an adequate alternative for the treatment of domestic wastewater in small communities, although it warns of the need for regular monitoring and adjustment of treatment conditions, especially during cooler periods.
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