Bioaugmentation and conjugative plasmid transference using bacteria from extreme environments to enhance biodegradation of recalcitrant pollutants in WWTP granular sludge

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Batch studies on the biodegradation potential of Paracetamol, Fluoxetine and 17α-Ethinylestradiol by the Micrococcus yunnanensis Strain TJPT4 recovered from marine organisms

Publication . Da Luz Palma, Tânia Cristina; Valentine, Julia; Gomes, Vera; Faleiro, L; Costa, Maria

The emerging pollutants paracetamol, fluoxetine and ethinylestradiol are inefficiently removed by conventional wastewater treatments, entering in aquatic environments in which they are hazardous. Aiming for the obtention of bacteria with the capacity for environmental bioremediation, eight bacteria were isolated from two consortia recovered from Hymedesmia versicolor and Filograna implexa marine organisms which exhibited a high-paracetamol-removal capacity. The isolates that displayed the ability to grow in the presence of 100 mg/L paracetamol as the sole carbon source were assigned to Paenibacillus, Micrococcus and Microbacterium genera. The isolate assigned to the Micrococcus yunnanensis strain TJPT4 presented the best performance, degrading 93 ± 4% of 15 mg/L paracetamol as the sole carbon source after 360 h, and was also apparently able to degrade the produced metabolites. This strain was able to remove 82.1 ± 0.9% of 16 mg/L fluoxetine after 504 h, mainly by adsorption, but apparently a biodegradation contribution also occurred. This strain was able to remove 66.6 ± 0.2% of 13 mg/L 17α-ethinylestradiol after 360 h. As far as is known, Micrococcus yunnanensis is for the first time recovered/identified in Filograna implexa, presenting a high drug removal efficiency, thereby becoming a great candidate for treatment processes (e.g., bioaugmentation), especially in the presence of saline intrusions.

2022-10-23Journal article

Open Access

Biodegradation of paracetamol and its intermediate metabolite hydroquinone by bacterial strains isolated from two mines of the iberian pyrite belt

Publication . Ismail, Fatma M.A. Elsayed; Costa, Maria Clara; Carlier, Jorge Dias

The main objective of the current study was to isolate bacterial strains able to biodegrade the emerging pollutants paracetamol (APAP) and hydroquinone (HQ), amongst the most worldwide prescribed drugs, also frequently detected in wastewater treatment plants influents and effluents and the environment. The most promising microbial consortia of Poderosa and Lousal mines for APAP removal were selected based on the previous reports of PROBIOMA project (European Regional Development Fund ERDF - Interreg V-A Spain-Portugal program (POCTEP)). The ability of the selected microbial consortia to remove APAP from the Mineral Salt Medium (MSM) at an initial concentration of about 500 mgL-1 (MSM-APAP (500 mgL-1)), under dark shaking conditions of 160 rpm at 25 °C, was confirmed using UV-vis molecular spectroscopy. Subsequently, the isolation step from selected samples proceeded from three successive enrichment cultures using MSM-drug (500 mgL-1) under the aforementioned conditions by spreading first on LB-drug and then on MSM-drug (500 mgL-1) agar plates and resulted in seven isolates able to utilize APAP as sole carbon source, and identified according to 16S rRNA gene sequence analysis as members of genera Aeromonas, Bacillus (two isolates), Niallia, Paraburkholderia, Rhizobium, and Variovorax, as well as one HQ utilizing isolate (Mycolicibacterium sp.). The HPLC analysis of APAP removal, in MSM-APAP (500 mgL-1) under the same culture conditions, by the two putative APAP biodegrading Bacillus sp. isolates revealed that Bacillus sp. (PDE3.1) showed maximal APAP %removal of 63+3 after 18 days, while Bacillus. sp. (PLC2.1) showed %removal of only 8+1 at the end of the experiment after 21 days. The key metabolites of APAP degradation (4-aminophenol and HQ) were detected through GC-MS analysis in the experiment with Bacillus sp. (PDE3.1) at very low concentrations. Then, the seven potential APAP biodegrading bacterial isolates were tested for APAP removal in MSM at a lower concentration of 50 mgL-1. Rhizobium sp. (PDE3.3) and Paraburkholderia sp. (PLA3.3) seemed the most promising where APAP %removal was 49±4 and 47.9±0.9, respectively. Later, the co-culture of the three most promising isolates (Rhizobium sp. (PDE3.3), Paraburkholderia sp. (PLA3.3), and Bacillus sp. (PDE3.1) didn’t improve the %removal compared with the pure cultures, while the co-culture with the seven potential APAP biodegrading isolates did not show removal capacity. Mycolicibacterium sp. (HPB1.1) showed at least 88% removal of HQ from MSM-HQ (50 mgL-1) after four days; hence, was checked for APAP removal in MSM-APAP (50 mgL-1) and showed APAP %removal of 41.6±0.1. Overall, some bacterial strains isolated from Poderosa and Lousal mines showed removal capacity; hence, more efforts should be directed at investigating if biodegradation is the main removal mechanism involved, and at exploring the biodegradation potentials of The Iberian Pyrite Belt mines associated bacteria.

2022-12-12Master thesis

Open Access

Implementation of standard methods for parameters routinely monitored in wastewater treatment plants and application in bioaugmentation with mycolicibacterium sp. In granular sludge reactors

Publication . Igbodo, Benjamin; Costa, Maria Clara; Carliar, Jorge

The effective treatment of domestic wastewater is pivotal for the health and sanitation status of water resources and wastewater reclamation campaigns. Thus, this study was aimed at evaluating the performance of Aerobic granular Sludge (AGS) in the treatment of synthetic wastewater during a bacteria bioaugmentation (Mycolicibacterium spp strain) experiment using laboratory-scale Sequencing Batch Reactors (SBRs). The routine parameters evaluated using UV–visible spectrophotometry methods were Total Nitrogen (TN), ammonia nitrogen (NH3 - N), nitrate nitrogen (NO3 - –N), phosphate phosphorus (PO4 3- - P) and Chemical Oxygen Demand (COD), on samples from the influents and effluents of the SBRs. The routine parameters evaluated using UV–visible spectrophotometry methods were Total Nitrogen (TN), ammonia nitrogen (NH3 - N), nitrate nitrogen (NO3- – N), phosphate phosphorus (PO43- - P) and Chemical Oxygen Demand (COD), on samples from the influents and effluents of the SBRs. Nitrite nitrogen (NO2- - N) was evaluated by mass balance and pH was measured using a benchtop pH meter. The individual standard calibration curves for the parameters had regression coefficients > 0.995 and the LOD (for qualitative analysis) and LOQ (for quantitative analysis) values were compatible with the concentrations of the samples analyzed while the validation process (using reference materials) yielded relative accuracy > 94 %.

2023-09-15Master thesis

Open Access

Effect of composting on biodegradation of compostable and conventional microplastic by bacterial communities from sewage sludge

Publication . Alcivar, Adriana Rosaura Gonzalez; Beltrán, Isabel Marín; Costa, Maria Clara

Microplastics (MP) threaten the aquatic and terrestrial ecosystems. Several studies have reported that wastewater treatment plants (WWTPs) are one of the main sources of MP to the ocean, even though they remove up to 98% of MPs from the influent (Carr et al., 2016; Murphy et al., 2016; Simon et al., 2018; Talvitie et al., 2017). Most of these MPs are not really eliminated but end up in the sewage sludge. This sludge is normally used as fertilizer in agricultural soils, after a composting process. However, current regulation does not consider the concentration of MP in sludge before their application on natural soils. This work focused on evaluating the effect of composting on MP particles. For this, we spiked sewage sludge from a European WWTP with MP films (2 mm-2 ) from a polyethylene terephthalate (PET) bottle and a compostable (according to the seller) bag of polyethylene (CPE). The experimental set-up consisted of: sludge spiked with 40 PET MP, sludge spiked with 40 CPE MP. These two treatments were repeated but inoculating a bacterial isolate from the genus Bacillus. Sludge with no added MP or bacterial inoculum was used as control. Samples were kept at 50ºC for 60 days, and organic matter content, pH, C:N ratio, the change in the functional groups of MP, and changes in the bacterial community were monitored monthly. Results showed that 40 MP had no effect on the composting process. There were differences among peaks indicating chemical changes and biodegradation for PET and CPE MP. After 60 days, scanning electron microscopy revealed adhering biofilms and a hole for CPE MP submitted to bioaugmentation treatment but did not decompose as expected. The concentration of MPs in the sludge after 60 days was 27 MP by g-1 , compared to 4 MP by g-1 at the beginning. Polyester (28%) was the most abundant polymer and fibers (84%) the most common shape.

2023-09-21Master thesis

Embargo

Batch studies on the biodegradation of paracetamol and 1,4-hydroquinone by novel bacterial strains isolated from extreme environmental samples and the identification of candidate catabolic genes

Publication . Lara-Moreno, Alba; Fatma El-Sayed; Cox, Cymon; Costa, Maria Clara; Carlier, Jorge

The emerging pollutant paracetamol (APAP) is one of the most prescribed drugs worldwide. In addition, APAP and its main metabolites, namely, 4-aminophenol (4-AP), hydroquinone (H2Q), benzoquinone (BQ), and 2,5-dihydroxy-1,4-benzoquinone (2,5-OH-BQ), among others, are frequently detected in wastewater treatment plants (WWTPs) influents, effluents, and the environment. Thus, continuous release into the environment, especially aquatic environments, is a source of general concern. Six APAP-degrading bacterial strains were isolated from two mine samples from the Iberian Pyrite Belt (Lousal and Poderosa mines). Mycolicibacterium aubagnense HPB1.1, which was isolated using enrichment cultures from the Poderosa mine sample in the presence of H2Q as the sole carbon source, also showed APAP biodegrading capabilities. Pure cultures of this strain degraded 34.3 mg L-1 of APAP in 5 days and 9.4 mg L-1 of H2Q in 4 days. Interestingly, BQ and 2,5-OH-BQ were detected as metabolites resulting from H2Q abiotic degradation, but these compounds were removed in the strain's cultures. Furthermore, M. aubagnense HPB1.1 whole-genome was sequenced, and its encoded proteins were aligned with enzymes of APAP-degrading bacteria recovered from databases and literature aiming to identify candidate catabolic genes. Putative amidases, deaminases, hydroxylases, and dioxygenases, responsible for the degradation of APAP by the HPB1.1 strain, were identified by similarity, corroborating its ability to transform APAP and its intermediate metabolite H2Q into less toxic metabolic compounds due to their capacity to break the aromatic ring of these molecules.

2024-08-13Journal article

Open Access