Substrate pH mediates growth promotion and resilience to water stress of Tilia tomentosa seedlings after Ectomycorrhizal inoculation.

Colonization by Ectomycorrhizal (EcM) fungi is key for the health and performance of plants under different stress scenarios, such as those faced by trees in urban environments. Because urban environments can be lacking EcM fungi, we here assessed the benefits of inoculating Tilia tomentosa seedlings in a pre-transplantation nursery context with the EcM fungi Lactarius deliciosus and Paxillus involutus, using substrates of different pH and facing water-stress. P. involutus had a more evident positive effect in T. tomentosa seedlings and had a good performance in both acidic and alkaline substrate. In acidic substrate the fungus increased the plant height by 0.91-fold, increased the mycorrhization rate by 3.23-fold, expansion rate by 5.03-fold and formation of secondary roots by 0.46-fold, compared to the non-inoculated control. This species also improved the phosphorus content of leaves, which revealed a promotion of nutrient uptake. In alkaline substrate P. involutus increased root dry weight by 3.92-fold and the mycorrhization parameters. In contrast, L. deliciosus only had a positive effect in the improvement of mycorrhization and expansion rates and phosphorus content in the root, effects visible only in alkaline substrate. When exposed to water-stress the increase of proline content was visible in acidic substrate for both fungi, L. deliciosus and P. involutus, and in alkaline substrate for the fungus P. involutus, a response indicative of the enhancement of defenses in stressing scenarios such as water scarcity. We conclude that fungal inoculation improves the vigour and resilience of Tilia seedlings and that it is of utmost importance to select a suitable EcM fungus and to consider the soil pH of the transplanting site. The inoculation approach can be a valuable tool to produce robust seedlings which may have a better performance when transplanted to the challenging urban environment.

Enhancing Native Plant Establishment in Mine Tailings under Drought Stress Conditions through the Application of Organo-Mineral Amendments and Microbial Inoculants.

The implementation of phytoremediation strategies under arid and semiarid climates requires the use of appropriate plant species capable of withstanding multiple abiotic stresses. In this study, we assessed the combined effects of organo-mineral amendments and microbial inoculants on the chemical and biological properties of mine tailings, as well as on the growth of native plant species under drought stress conditions. Plants were cultivated in pots containing 1 kg of a mixture of mine tailings and topsoil (i.e., pre-mined superficial soil) in a 60:40 ratio, 6% marble sludge, and 10% sheep manure. Moreover, a consortium of four drought-resistant plant growth-promoting rhizobacteria (PGPR) was inoculated. Three irrigation levels were applied: well-watered, moderate water deficit, and severe water deficit, corresponding to 80%, 45%, and 30% of field capacity, respectively. The addition of topsoil and organo-mineral amendments to mine tailings significantly improved their chemical and biological properties, which were further enhanced by bacterial inoculation and plants' establishment. Water stress negatively impacted enzymatic activities in amended tailings, resulting in a significant decrease in acid and alkaline phosphatases, urease, and dehydrogenase activities. Similar results were obtained for bacteria, fungi, and actinomycete abundance. PGPR inoculation positively influenced the availability of phosphorus, total nitrogen, and organic carbon, while it increased alkaline phosphatase, urease (by about 10%), and dehydrogenase activity (by 50%). The rhizosphere of Peganum harmala showed the highest enzymatic activity and number of culturable microorganisms, especially in inoculated treatments. Severe water deficit negatively affected plant growth, leading to a 40% reduction in the shoot biomass of both Atriplex halimus and Pennisetum setaceum compared to well-watered plants. P. harmala showed greater tolerance to water stress, evidenced by lower decreases observed in root and shoot length and dry weight compared to well-watered plants. The use of bioinoculants mitigated the negative effects of drought on P. harmala shoot biomass, resulting in an increase of up to 75% in the aerial biomass in plants exposed to severe water deficit. In conclusion, the results suggest that the combination of organo-mineral amendments, PGPR inoculation, and P. harmala represents a promising approach to enhance the phytoremediation of metal-polluted soils under semiarid conditions.

Impact of soybean-associated plant growth-promoting bacteria on plant growth modulation under alkaline soil conditions.

Conventional strategies to manage iron (Fe) deficiency still present drawbacks, and more eco-sustainable solutions are needed. Knowledge on soybean-specific diversity and functional traits of their plant growth-promoting bacteria (PGPB) potentiates their applicability as bioinoculants to foster soybean performance under calcareous soil conditions. This work aimed to assess the efficacy of PGPB, retrieved from soybean tissues/rhizosphere, in enhancing plant growth and development as well as crop yield under alkaline soil conditions. Seventy-six bacterial strains were isolated from shoots (18%), roots (53%), and rhizosphere (29%) of soybean. Twenty-nine genera were identified, with Bacillus and Microbacterium being the most predominant. Based on distinct plant growth-promoting traits, the endophyte Bacillus licheniformis P2.3 and the rhizobacteria Bacillus aerius S2.14 were selected as bioinoculants. In vivo tests showed that soybean photosynthetic parameters, chlorophyll content, total fresh weight, and Fe concentrations were not significantly affected by bioinoculation. However, inoculation with B. licheniformis P2.3 increased pod number (33%) and the expression of Fe-related genes (FRO2, IRT1, F6'H1, bHLH38, and FER4), and decreased FC-R activity (45%). Moreover, bioinoculation significantly affected Mn, Zn, and Ca accumulation in plant tissues. Soybean harbors several bacterial strains in their tissues and in the rhizosphere with capacities related to Fe nutrition and plant growth promotion. The strain B. licheniformis P2.3 showed the best potential to be incorporated in bioinoculant formulations for enhancing soybean performance under alkaline soil conditions.

Bioremediation of coastal aquaculture effluents spiked with florfenicol using microalgae-based granular sludge - a promising solution for recirculating aquaculture systems.

Aquaculture is a crucial industry in the agri-food sector, but it is linked to serious environmental problems. There is a need for efficient treatment systems that allow water recirculation to mitigate pollution and water scarcity. This work aimed to evaluate the self-granulation process of a microalgae-based consortium and its capacity to bioremediate coastal aquaculture streams that sporadically contain the antibiotic florfenicol (FF). A photo-sequencing batch reactor was inoculated with an autochthonous phototrophic microbial consortium and was fed with wastewater mimicking coastal aquaculture streams. A rapid granulation process occurred within ca. 21 days, accompanied by a substantially increase of extracellular polymeric substances in the biomass. The developed microalgae-based granules exhibited high and stable organic carbon removal (83-100%). Sporadically wastewater contained FF which was partially removed (ca. 5.5-11.4%) from the effluent. In periods of FF load, the ammonium removal slightly decreased (from 100 to ca. 70%), recovering 2 days after FF feeding ceased. A high-chemical quality effluent was obtained, complying with ammonium, nitrite, and nitrate concentrations for water recirculation within a coastal aquaculture farm, even during FF feeding periods. Members belonging to the Chloroidium genus were predominant in the reactor inoculum (ca. 99%) but were replaced from day-22 onwards by an unidentified microalga from the phylum Chlorophyta (>61%). A bacterial community proliferated in the granules after reactor inoculation, whose composition varied in response to feeding conditions. Bacteria from the Muricauda and Filomicrobium genera, Rhizobiaceae, Balneolaceae, and Parvularculaceae families, thrived upon FF feeding. This study demonstrates the robustness of microalgae-based granular systems for aquaculture effluent bioremediation, even during periods of FF loading, highlighting their potential as a feasible and compact solution in recirculation aquaculture systems.

Biodegradation and Metabolic Pathway of the Neonicotinoid Insecticide Thiamethoxam by Labrys portucalensis F11.

Thiamethoxam (TMX) is an effective neonicotinoid insecticide. However, its widespread use is detrimental to non-targeted organisms and water systems. This study investigates the biodegradation of this insecticide by Labrys portucalensis F11. After 30 days of incubation in mineral salt medium, L. portucalensis F11 was able to remove 41%, 35% and 100% of a supplied amount of TMX (10.8 mg L-1) provided as the sole carbon and nitrogen source, the sole carbon and sulfur source and as the sole carbon source, respectively. Periodic feeding with sodium acetate as the supplementary carbon source resulted in faster degradation of TMX (10.8 mg L-1); more than 90% was removed in 3 days. The detection and identification of biodegradation intermediates was performed by UPLC-QTOF/MS/MS. The chemical structure of 12 metabolites is proposed. Nitro reduction, oxadiazine ring cleavage and dechlorination are the main degradation pathways proposed. After biodegradation, toxicity was removed as indicated using Aliivibrio fischeri and by assessing the synthesis of an inducible ß-galactosidase by an E. coli mutant (Toxi-Chromo test). L. portucalensis F11 was able to degrade TMX under different conditions and could be effective in bioremediation strategies.

Valorization of Brewery Waste through Polyhydroxyalkanoates Production Supported by a Metabolic Specialized Microbiome.

Raw brewers' spent grain (BSG), a by-product of beer production and produced at a large scale, presents a composition that has been shown to have potential as feedstock for several biological processes, such as polyhydroxyalkanoates (PHAs) production. Although the high interest in the PHA production from waste, the bioconversion of BSG into PHA using microbial mixed cultures (MMC) has not yet been explored. This study explored the feasibility to produce PHA from BSG through the enrichment of a mixed microbial culture in PHA-storing organisms. The increase in organic loading rate (OLR) was shown to have only a slight influence on the process performance, although a high selectivity in PHA-storing microorganisms accumulation was reached. The culture was enriched on various PHA-storing microorganisms, such as bacteria belonging to the Meganema, Carnobacterium, Leucobacter, and Paracocccus genera. The enrichment process led to specialization of the microbiome, but the high diversity in PHA-storing microorganisms could have contributed to the process stability and efficiency, allowing for achieving a maximum PHA content of 35.2 ± 5.5 wt.% (VSS basis) and a yield of 0.61 ± 0.09 CmmolPHA/CmmolVFA in the accumulation assays. Overall, the production of PHA from fermented BSG is a feasible process confirming the valorization potential of the feedstock through the production of added-value products.

Microalgae-bacterial biomass outperforms PN-anammox biomass for oxygen saving in continuous-flow granular reactors facing extremely low-strength freshwater aquaculture streams.

The dissolved oxygen (DO) concentration in water streams is one of the most important and critical quality parameters in aquaculture farms. The main objective of this study was to evaluate the potential of two Continuous Flow Granular Reactors, one based on Partial Nitrification-Anammox biomass (Aquammox CFGR) and the other on Microalgae-Bacteria biomass (AquaMab CFGR), for improving dissolved oxygen availability in the recirculation aquaculture systems (RAS). Both reactors treated the extremely low-strength effluents from a freshwater trout farm (1.39 mg NH4+-N/L and 7.7 mg TOC/L). The Aquammox CFGR, removed up to 68% and 100% of ammonium and nitrite, respectively, but the DO concentration in the effluent was below 1 mg O2/L while the anammox activity was not maintained. In the AquaMab CFGR, bioaugmentation of aerobic granules with microalgae was attained, producing an effluent with DO concentrations up to 9 mg O2/L and removed up to 77% and 80% of ammonium and nitrite, respectively, which is expected to reduce the aeration costs in fish farms.

Biodegradation and Metabolic Pathway of 17ß-Estradiol by Rhodococcus sp. ED55.

Endocrine disrupting compounds (EDCs) in the environment are considered a motif of concern, due to the widespread occurrence and potential adverse ecological and human health effects. The natural estrogen, 17ß-estradiol (E2), is frequently detected in receiving water bodies after not being efficiently removed in conventional wastewater treatment plants (WWTPs), promoting a negative impact for both the aquatic ecosystem and human health. In this study, the biodegradation of E2 by Rhodococcus sp. ED55, a bacterial strain isolated from sediments of a discharge point of WWTP in Coloane, Macau, was investigated. Rhodococcus sp. ED55 was able to completely degrade 5 mg/L of E2 in 4 h in a synthetic medium. A similar degradation pattern was observed when the bacterial strain was used in wastewater collected from a WWTP, where a significant improvement in the degradation of the compound occurred. The detection and identification of 17 metabolites was achieved by means of UPLC/ESI/HRMS, which proposed a degradation pathway of E2. The acute test with luminescent marine bacterium Aliivibrio fischeri revealed the elimination of the toxicity of the treated effluent and the standardized yeast estrogenic (S-YES) assay with the recombinant strain of Saccharomyces cerevisiae revealed a decrease in the estrogenic activity of wastewater samples after biodegradation.

Actinobacteria isolated from wastewater treatment plants located in the east-north of Algeria able to degrade pesticides.

The pollution of water resources by pesticides poses serious problems for public health and the environment. In this study, Actinobacteria strains were isolated from three wastewater treatment plants (WWTPs) and were screened for their ability to degrade 17 pesticide compounds. Preliminary screening of 13 of the isolates of Actinobacteria allowed the selection of 12 strains with potential for the degradation of nine different pesticides as sole carbon source, including aliette, for which there are no previous reports of biodegradation. Evaluation of the bacterial growth and degradation kinetics of the pesticides 2,4-dichlorophenol (2,4-DCP) and thiamethoxam (tiam) by selected Actinobacteria strains was performed in liquid media. Strains Streptomyces sp. ML and Streptomyces sp. OV were able to degrade 45% of 2,4-DCP (50 mg/l) as the sole carbon source in 30 days and 84% of thiamethoxam (35 mg/l) in the presence of 10 mM of glucose in 18 days. The biodegradation of thiamethoxam by Actinobacteria strains was reported for the first time in this study. These strains are promising for use in bioremediation of ecosystems polluted by this type of pesticides.

Biological removal processes in aerobic granular sludge exposed to diclofenac.

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.

Cultivable microalgae diversity from a freshwater aquaculture filtering system and its potential for polishing aquaculture-derived water streams.

AIMS: Microalgae are ubiquitous in aquatic environments, including aquaculture farms, but few studies have delved into their phytoplankton diversity and bioremediation potential. In this study, the cultivable phytoplankton of a rainbow trout freshwater aquaculture farm was isolated, phylogenetically analysed and used to assemble a consortium to polish an aquaculture-derived effluent, with low concentrations of ammonium, nitrite and nitrate. METHODS AND RESULTS: Through standard plating in different selective media, a total of 15 microalgae strains were isolated from sludge from a rotary drum filtering system which removes suspended solids from the water exiting the facility. Based on 18S rRNA gene sequences, isolates were assigned to nine different genera of the Chlorophyta phylum: Asterarcys, Chlorella, Chloroccocum, Chlorosarcinopsis, Coelastrella, Desmodesmus, Micractinium, Parachlorella and Scenedesmus. Species from most of these genera are known to inhabit freshwater systems in Galicia and continental Spain, but the Coelastrella, Asterarcys or Parachlorella genera are not usually present in freshwater streams. In an onsite integrative approach, the capacity of a consortium of native microalgae isolates to grow on aquaculture-derived effluents and its nutrient removal capacity were assessed using a raceway pond. After 7 days, removal efficiencies of approximately 99%, 92% and 49% for ammonium, nitrite and nitrate, respectively, were achieved concomitantly with a microalgae biomass increase of ca. 17%. CONCLUSIONS: Sludge from the aquaculture filtering system presents a high diversity of microalgae species from the Chlorophyta phylum, whose application in a consortial approach revealed to be efficient to polish aquaculture-derived effluents with low nutrient content. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of native microalgae consortia from aquaculture systems can contribute to the development of efficient treatment systems for low-nutrient wastewater, avoiding nutrients release to the environment and promoting water recirculation. This may further strengthen the use of phycoremediation at the industrial scale, as an environment-friendly strategy.

Quantitative image analysis as a robust tool to assess effluent quality from an aerobic granular sludge system treating industrial wastewater.

Quantitative image analysis (QIA) is a simple and automated method for process monitoring, complementary to chemical analysis, that when coupled to mathematical modelling allows associating changes in the biomass to several operational parameters. The majority of the research regarding the use of QIA has been carried out using synthetic wastewater and applied to activated sludge systems, while there is still a lack of knowledge regarding the application of QIA in the monitoring of aerobic granular sludge (AGS) systems. In this work, chemical oxygen demand (COD), ammonium (N-NH4+), nitrite (N-NO2-), nitrate (N-NO3-), salinity (Cl-), and total suspended solids (TSS) levels present in the effluent of an AGS system treating fish canning wastewater were successfully associated to QIA data, from both suspended and granular biomass fractions by partial least squares models. The correlation between physical-chemical parameters and QIA data allowed obtaining good assessment results for COD (R2 of 0.94), N-NH4+ (R2 of 0.98), N-NO2- (R2 of 0.96), N-NO3- (R2 of 0.95), Cl- (R2 of 0.98), and TSS (R2 of 0.94). While the COD and N-NO2- assessment models were mostly correlated to the granular fraction QIA data, the suspended fraction was highly relevant for N-NH4+ assessment. The N-NO3-, Cl- and TSS assessment benefited from the use of both biomass fractions (suspended and granular) QIA data, indicating the importance of the balance between the suspended and granular fractions in AGS systems and its analysis. This study provides a complementary approach to assess effluent quality parameters which can improve wastewater treatment plants monitoring and control, with a more cost-effective and environmentally friendly procedure, while avoiding daily physical-chemical analysis.

Sequencing versus continuous granular sludge reactor for the treatment of freshwater aquaculture effluents.

Ammonium and nitrite levels in water are crucial for fish health preservation and growth maintenance in freshwater aquaculture farms, limiting water recirculation. The aim of the present work was the evaluation and comparison of two granular sludge reactors which were operated to treat freshwater aquaculture streams at laboratory-scale: an Aerobic Granular Sludge - Sequencing Batch Reactor (AGS-SBR) and a Continuous Flow Granular Reactor (CFGR). Both units were fed with a synthetic medium mimicking an aquaculture recycling water (1.9-2.9 mg N/L), with low carbon content, and operational temperature varied between 17 and 25 °C. The AGS-SBR, inoculated with mature granules from a full-scale wastewater treatment plant, achieved high carbon and ammonium removal during the 157 operational days. Even at low hydraulic retention time (HRT), varying from 474 to 237 min, ammonium removal efficiencies of approximately 87-100% were observed, with an ammonium removal rate of approximately 14.5 mg NH4+-N/(L⋅d). Partial biomass washout occurred due to the extremely low carbon and nitrogen concentrations in the feeding, which could only support the growth of a small portion of bacteria, but no major changes on the reactor removal performance were observed. The CFGR was inoculated with activated sludge and operated for 98 days. Biomass granulation occurred in 7 days, improving the settling properties due to a high up-flow velocity of 11 m/h and an applied HRT of 5 min. The reactor presented mature granules after 32 days, achieving an average diameter of 1.9 mm at day 63. The CFGR ammonium removal efficiencies were of approximately 10-20%, with ammonium removal rates of 90.0 mg NH4+-N/(L⋅d). The main biological processes taking place in the AGS-SBR were nitrification and heterotrophic growth, while in the CFGR the ammonium removal occurred only by heterotrophic assimilation, with the reactor also presenting complete and partial denitrification, which caused nitrite production. Comparing both systems, the CFGR achieved 6 times higher ammonium removal rates than the AGS-SBR, being suitable for treating extremely high flows. On the other hand, the AGS-SBR removed almost 100% of ammonium content in the wastewater, discharging a better quality effluent, less toxic for the fish but treated lower flows.

Sediments in the mangrove areas contribute to the removal of endocrine disrupting chemicals in coastal sediments of Macau SAR, China, and harbour microbial communities capable of degrading E2, EE2, BPA and BPS.

The occurrence of endocrine disrupting chemicals (EDCs) is a major issue for marine and coastal environments in the proximity of urban areas. The occurrence of EDCs in the Pearl River Delta region is well documented but specific data related to Macao is unavailable. The levels of bisphenol-A (BPA), estrone (E1), 17α-estradiol (αE2), 17ß-estradiol (E2), estriol (E3), and 17α-ethynylestradiol (EE2) were measured in sediment samples collected along the coastline of Macao. BPA was found in all 45 collected samples with lower BPA concentrations associated to the presence of mangrove trees. Biodegradation assays were performed to evaluate the capacity of the microbial communities of the surveyed ecosystems to degrade BPA and its analogue BPS. Using sediments collected at a WWTP discharge point as inoculum, at a concentration of 2 mg l-1 complete removal of BPA was observed within 6 days, whereas for the same concentration BPS removal was of 95% after 10 days, which is particularly interesting since this compound is considered recalcitrant to biodegradation and likely to accumulate in the environment. Supplementation with BPA improved the degradation of bisphenol-S (BPS). Aiming at the isolation of EDCs-degrading bacteria, enrichments were established with sediments supplied with BPA, BPS, E2 and EE2, which led to the isolation of a bacterial strain, identified as Rhodoccoccus sp. ED55, able to degrade the four compounds at different extents. The isolated strain represents a valuable candidate for bioremediation of contaminated soils and waters.

Simultaneous nitrification and phosphate removal by bioaugmented aerobic granules treating a fluoroorganic compound.

The presence of toxic compounds in wastewater can cause problems for organic matter and nutrient removal. In this study, the long-term effect of a model xenobiotic, 2-fluorophenol (2-FP), on ammonia-oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and phosphate accumulating organisms (PAO) in aerobic granular sludge was investigated. Phosphate (P) and ammonium (N) removal efficiencies were high (>93%) and, after bioaugmentation with 2-FP degrading strain FP1, 2-FP was completely degraded. Neither N nor P removal were affected by 50 mg L-1 of 2-FP in the feed stream. Changes in the aerobic granule bacterial communities were followed. Numerical analysis of the denaturing gradient gel electrophoresis (DGGE) profiles showed low diversity for the ammonia monooxygenase (amoA) gene with an even distribution of species. PAOs, including denitrifying PAO (dPAO), and AOB were present in the 2-FP degrading granules, although dPAO population decreased throughout the 444 days reactor operation. The results demonstrated that the aerobic granules bioaugmented with FP1 strain successfully removed N, P and 2-FP simultaneously.

Assessment of an aerobic granular sludge system in the presence of pharmaceutically active compounds by quantitative image analysis and chemometric techniques.

In this study, a sequencing batch reactor (SBR) with aerobic granular sludge (AGS) was operated with synthetic wastewater containing environmental relevant concentrations of 17ß-estradiol (E2), 17α-ethinylestradiol (EE2) and sulfamethoxazole (SMX). Despite the presence of the studied PhAC, the granular fraction clearly predominated (TSSgran/TSS ranging from 0.82 to 0.98) throughout the monitoring period, presenting aggregates with high organic fraction (VSS/TSS above 0.83) and good settling characteristics (SVI5 ranging from 15 to 39 mL/gTSS). A principal component analysis (PCA) with quantitative image analysis (QIA) based data allowed to distinguish the different operational periods, namely with mature granules (CONT), and the E2, EE2, and SMX feeding periods. It further revealed a positive relationship between the biomass density, sludge settling ability, overall and granular biomass contents, granulation properties, granular biomass fraction and large granules fraction and size. Moreover, a discriminant analysis (DA) allowed to successfully discriminate not only the different operational periods, mainly by using the floccular apparent density, granular stratification and contents data, but also the PhAC presence in samples. The filamentous bacteria contents, sludge settling properties, settling properties stability and granular stratification, structure and contents parameters were found to be crucial for that purpose.

Recovered granular sludge extracellular polymeric substances as carrier for bioaugmentation of granular sludge reactor.

An increasing amount of industrial chemicals are being released into wastewater collection systems and indigenous microbial communities in treatment plants are not always effective for their removal. In this work, extracellular polymeric substances (EPS) recovered from aerobic granular sludge (AGS) were used as a natural carrier to immobilize a specific microbial strain, Rhodococcus sp. FP1, able to degrade 2-fluorophenol (2-FP). The produced EPS granules exhibited a 2-FP degrading ability of 100% in batch assays, retaining their original activity after up to 2-months storage. Furthermore, EPS granules were added to an AGS reactor intermittently fed with saline wastewater containing 2-FP. Degradation of 2-FP and stoichiometric fluorine release occurred 8 and 35 days after bioaugmentation, respectively. Chemical oxygen demand removal was not significantly impaired by 2-FP or salinity loads. Nutrients removal was impaired by 2-FP load, but after bioaugmentation, the phosphate and ammonium removal efficiency improved from 14 to 46% and from 25 to 42%, respectively. After 2-FP feeding ceased, at low/moderate salinity (0.6-6.0 g L-1 NaCl), ammonium removal was completely restored, and phosphate removal efficiency increased. After bioaugmentation, 11 bacteria isolated from AGS were able to degrade 2-FP, indicating that horizontal gene transfer could have occurred in the reactor. The improvement of bioreactor performance after bioaugmentation with EPS immobilized bacteria and the maintenance of cell viability through storage are the main advantages of the use of this natural microbial carrier for bioaugmentation, which can benefit wastewater treatment processes.

Long-term stability of a non-adapted aerobic granular sludge process treating fish canning wastewater associated to EPS producers in the core microbiome.

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: CandidatusAccumulibacter, 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.

Quantification of fluoroquinolones in wastewaters by liquid chromatography-tandem mass spectrometry.

Antibiotics are the most consumed therapeutic classes worldwide and are released to the environment in their original form as well as potentially active metabolites and/or degradation products. Consequences of the occurrence of these compounds in the environment are primarily related to bacterial resistance development. This work presents a validated analytical method based on solid phase extraction (SPE) using HLB cartridges, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of seven different fluoroquinolone antibiotics, namely ciprofloxacin (CPF), enrofloxacin (ENR), lomefloxacin (LOM), norfloxacin (NOR), ofloxacin (OFL), prulifloxacin (PLF) and moxifloxacin (MOX) and its application to detect the target compounds in influents and effluents of wastewater treatment plants (WWTP). Linearity was established through calibration curves in solvent and matrix match using internal calibration method in the range of 50-1300 ng L-1 and all the fluoroquinolones showed good linear fit (r2 ≥ 0.991). Accuracy ranged between 80.3 and 92.9%, precision was comprised between 7.2 and 14.6%, and 10.7 and 18.1% for intra- and inter-batch determinations, respectively. Method detection and quantification limits ranged from 6.7 to 59.0 ng L-1 and 22.3-196.6 ng L-1, respectively. Influents and effluents of fifteen WWTPs of North of Portugal were analyzed. OFL was the fluoroquinolone found at the highest concentration, up to 4587.0 ng L-1 and 987.9 ng L-1, in influent and effluent, respectively. NOR and PLF were not detected.