Degradation studies of widely used pharmaceutical compounds by heterogeneous photocatalysis and bacterial communities and isolates

The present thesis was focused on degradation studies of five extensively and globally used pharmaceutical compounds: chloramphenicol, paracetamol, fluoxetine and 17α-ethynilestradiol which are considered emerging pollutants that enter in the environment due to its inefficient removal by the conventional wastewater treatments plants. Photodegradation and biodegradation assays were realized. The photodegradation of chloramphenicol and paracetamol was successfully performed by heterogeneous photocatalysis using PbS/TiO2 nanocomposites, as catalysts, which were synthesized using biological sulphide produced by sulphate reducing bacteria in batch cultures. Although photodegradation demonstrated to be a faster process, biodegradation is preferable since it occurs without the use of external compounds, such as catalysts, using only the microorganisms already present in the environment. Communities and bacterial isolates with ability to remove and use these drugs as unique carbon sources were obtained using sludge collected from an oxidation ditch and a lagoon system of WWTPs. An initial aerobic bacterial community suffered a shift to favor the genera Pseudomonas sp; Flavobacterium, Dokdonella and Methylophilus which displayed high efficiency in removing 50 mg/L paracetamol as sole carbon source and are described for the first time as paracetamol degraders. Bacteria capable of effectively biodegrade paracetamol were isolated in aerobic conditions, suggesting for the first time that members of “Bacillus cereus group”, [Brevibacterium] frigoritolerans, Corynebacterium nuruki and Enterococcus faecium can use paracetamol as carbon source, whereas the isolates obtained for fluoxetine and 17α-ethynilestradiol only partially removed these drugs. An anaerobic consortium which was able to degrade 20 mg/L of FLX, as unique carbon source, to values below the limit of detection was obtained. The final consortium was mainly composed by genera vadinBC27 wastewater-sludge group, Macellibacteroidetes, Dethiosulfovibrio, Bacteroides, Tolumonas, Sulfuricurvum, f_Enterobacteriaceae_OTU_18, which are for the first time assumed as FLX biodegraders. In the biodegradation of 20 mg/L FLX, a possible metabolite, 2,3-dimethyl-5-(trifluoromethyl) benzene-1,4-diol, was detected after 28 of assay. Several attempts to optimize an aerobic granular system were performed to degrade ibuprofen. This process is still ongoing.A presente tese foca-se em estudos de degradação de 5 fármacos considerados poluentes emergentes, são estes: cloranfenicol, paracetamol, fluoxetina, 17α-etinilestradiol e ibuprofeno. Estes são considerados potenciais ameaças aos ecossistemas ambientais, à saúde e à segurança humana, especialmente devido ao seu uso extensivo a nível mundial. Os tratamentos de águas residuais convencionais têm-se mostrado ineficientes na remoção total destes fármacos resultando na sua libertação para o ambiente. Atualmente, uma das maiores preocupações é a contaminação incessante dos ambientes aquáticos com estes poluentes e os problemas ecológicos inerentes. Com o objetivo de conhecer, desenvolver ou melhorar os processos já existentes, foram realizados estudos de fotodegradação e biodegradação visando encontrar novas alternativas que sejam baratas e simples e que permitam uma completa remoção destes fármacos nas estações de tratamento de águas residuais (ETARs). Nos estudos de fotodegradação foram sintetizados com sucesso nanocompósitos de PbS/TiO2 (fotocatalisador heterogéneo) utilizando sulfureto produzido biologicamente pelas bactérias sulfato redutoras (BSR) em batch. A importância deste processo está no aproveitamento de resíduos tóxicos, como o sulfureto e iões chumbo em solução aquosa, para formulação de um catalisador eficiente na remoção destes poluentes (fármacos). Estes nanocompósitos, foram empregues com êxito como catalisadores na remoção de cloranfenicol e paracetamol, revelando uma elevada eficiência de remoção (>93%), após 240 minutos de irradiação. Nos ensaios de biodegradação obtiveram-se comunidades e isolados bacterianos com capacidade de utilizar os fármacos em estudo, como única fonte de carbono e que são descritos pela primeira vez como tendo um papel muito importante na remoção destes poluentes. Identificou-se uma comunidade bacteriana aeróbia que apresentou elevada eficácia na remoção de paracetamol como única fonte de carbono (97 ± 3%). Ao longo do ensaio verificou-se um “shift” na população inicial levando a um favorecimento dos géneros Pseudomonas sp; Flavobacterium, Dokdonella e Methylophilus, revelando pela primeira vez o seu possível envolvimento na degradação do paracetamol. Os metabolitos identificados nesse processo foram 4-aminofenol, hidroquinona e um composto desconhecido. Isolaram-se bactérias com capacidade de biodegradar paracetamol utilizando como inóculo as lamas recolhidas numa vala de oxidação, sugerindo pela primeira vez que membros de “Bacillus cereus group” (associados Bacillus pacificus ou Bacillus paranthracis), [Brevibacterium] frigoritolerans, Corynebacterium nuruki e Enterococcus faecium têm a capacidade de usar este fármaco como única fonte de carbono. Uma espécie pertencente ao “Bacillus cereus group” degradou 200 mg/L de paracetamol, abaixo dos níveis de deteção, sem formação aparente de metabolitos secundários tóxicos e removeu cerca de 95 ± 5% de 500 mg/L do fármaco, após 144 h de incubação a 28 ºC. Durante o processo de degradação do paracetamol foram detetados os metabolitos 4-aminofenol, hidroquinona e ácido 2-hexenóico. Realizaram-se estudos de biodegradação com uma comunidade bacteriana enriquecida a partir de uma amostra de lama recolhida num sistema de lagunagem em que foram favorecidas as BSR. As culturas foram efectuadas na presença de fluoxetina e lactato e usando o fármaco como única fonte de carbono, em condições redutoras e anaerobiose. O consórcio obtido revelou ter capacidade de degradar 20 mg/L e 50 mg/L de fluoxetina para valores abaixo do limite de deteção, após 28 e 31 dias, respetivamente. Utilizando fluoxetina como única fonte de carbono, verificou-se que o consórcio removeu 20 mg/L de fluoxetina abaixo dos limites de deteção e 66 ± 9% de 50 mg/L do fármaco, após 31 dias. Norfluoxetina foi identificada na degradação de 50 mg/L fluoxetina, enquanto na biodegradação de 20 mg/L de fluoxetina, foi detetado outro possível metabolito, o 2,3-dimetil-5-(trifluorometil)benzeno-1,4-diol, após 28 dias de ensaio. A população bacteriana inicial era constituída principalmente por Desulfomicrobium e Desulfovibrio, enquanto que no decurso das experiências usando fluoxetina como única fonte de carbono, verificou-se a ocorrência de um claro “shift” na comunidade com o aumento do grupo Wastewater-sludge group vadinBC27, Macellibacteroidetes, Dethiosulfovibrio, Bacteroides, Tolumonas, Sulfuricurvum, f_Enterobacteriaceae, bactérias identificadas pela primeira vez como possíveis degradadoras de fluoxetina. Em aerobiose, foram preparadas culturas inoculando o efluente bruto com a lama, simulando as condições da ETAR. Neste ensaio a remoção de fluoxetina foi de aproximadamente 89%, aparentemente com uma contribuição mínima de adsorção à lama. Durante este ensaio, a população inicial sofreu um “shift" com o aumento de estirpes dos géneros Aeromonadales_OTU_14, Acinetobacter, Rheinheimera, Shewanella, Pseudomonas, f_Pseudomonadaceae_OTU_4914; OTU_24; f_JI49D030_OTU_6171, Methylobacillus e Piscinobacter, sendo apontados pela primeira vez como os prováveis responsáveis pela biodegradação da fluoxetina. Da lama onde se obteve a comunidade anterior foram selecionados seis isolados com capacidade de utilizar fluoxetina como única fonte de carbono que foram identificados como Pseudomonas putida, Enterobacter ludwigii, Pseudomonas nitritireducens, Alcaligenes faecalis, Pseudomonas aeruginosa e Pseudomonas nitroreducens. Pseudomonas nitroreducens foi a mais eficiente, com remoções de 55 ± 1% e 21 ± 1% a 20 mg/L e 50 mg/L de fluoxetina, respetivamente, após 168 h. Os isolados obtidos não apresentaram elevadas percentagens de remoção deste fármaco, contudo estas foram significativas quando comparadas com o controle negativo. Embora o principal mecanismo de remoção de fluoxetina descrito na literatura seja por adsorção, neste estudo a biodegradação parece desempenhar o papel principal na degradação do fármaco. Das lamas ativadas foram obtidos isolados bacterianos com capacidade de crescimento na presença de 50 mg/L de 17α-etinilestradiol a partir de uma lama recolhida na vala de oxidação da ETAR Noroeste de Faro, sendo estes identificadas como Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans e Shinella zoogloeoides. Os resultados revelaram que Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans e Shinella zoogloeoides, degradaram 47 ± 4%, 55 ± 3%, 64 ± 4% e 35 ± 4%, respetivamente de 13 mg/L 17α-etinilestradiol, após 168 h a 28 ºC. A degradação de 17α-etinilestradiol por estes isolados resultou em vários intermediários/vias possíveis que são mais facilmente degradados e menos tóxicos do que o composto original. Os compostos detetados foram: ácido manónico 1,4-lactona pertencente às estruturas “gama lactona”, ácido 4-hidroxi-2-quinolinacarboxílico, ácido 2,3-di-hidroxibenzóico, ácido D-glucónico e ácido Z-aconítico, ácido 2-pentenodióico (ácido insaturado) e o ácido 2-butenodióico após possível meta-clivagem do anel A. O ácido 2-butenodióico (ácido fumárico), é um ácido dicarboxílico e um metabolito intermediário no ciclo dos ácidos tricarboxílicos/ciclo de Krebs. Em relação ao sistema granular realizaram-se diferentes abordagens a nível experimental visando optimizar o sistema e a degradação do ibuprofeno. Este processo ainda está em curso

Putative role of flavobacterium, dokdonella and methylophilus strains in paracetamol biodegradation

Paracetamol, the most widely and globally used analgesic and antipyretic, is easily accumulated in aquatic environments. In the present study, the biodegradation of paracetamol in different media (one for general growth, one specific for sulfate reducing bacteria, a mineral salts medium and municipal wastewater) inoculated with two types of sludge (from anaerobic lagoon and from oxidation ditch) under different oxygenic conditions (anoxic; moderate oxygenation in open flasks and high oxygenation by aeration) was investigated. In addition, bacteria with relative abundances increasing simultaneously with paracetamol degradation, when this drug was the only carbon source, thus with a putative role in its degradation, were identified using 16S rRNA gene sequences. The results show that aerobic microorganisms had a major role in the degradation of paracetamol, with 50 mg/L totally removed from municipal wastewater after 2 days incubation with aeration, and that the metabolites 4-aminophenol and hydroquinone plus one compound not identified in this work were produced in the process. The identification of bacteria with a role in the degradation of paracetamol revealed a strain from genus Pseudomonas with the highest final relative abundance of 21.2%, confirming previous works reporting strains of this genus as paracetamol decomposers. Besides, genera Flavobacterium, Dokdonella and Methylophilus were also in evidence, with initial relative abundances of 1.66%, 1.48 and 0.00% (not detected) in the inoculum and 6.91%, 3.80 and 3.83% after incubation, respectively. Therefore, a putative role of these genera in paracetamol biodegradation is suggested for the first time.Erasmus Mundus Master in Chemical Innovation and Regulations (EMMC-ChIR) of Donaldben Mbagag NEBA by the Erasmus Mundus program of the European Commissioninfo:eu-repo/semantics/publishedVersio

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

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.info:eu-repo/semantics/publishedVersio

Biodegradation of paracetamol by some gram-positive bacterial isolates

Bacterial isolates with the capacity to remove paracetamol were selected from an activated sludge sample collected in an oxidation ditch of a wastewater treatment plant. Among these, twelve bacterial isolates were selected according to their capacity to grow in the presence of paracetamol. They were identified using the colony morphotype procedure and by 16S rRNA gene sequencing analysis, but only four of them showed the ability to utilise paracetamol as the sole carbon source in the presence of a nitrogen supply. Those four bacterial isolates were assigned to species of the genera Bacillus, [Brevibacterium], Corynebacterium and Enterococcus. Bacterial isolates were cultured in liquid mineral salt medium (MSM) spiked with 200 mg/L of paracetamol at 28 °C in the dark. In cultures inoculated with [Brevibacterium] frigoritolerans, Corynebacterium nuruki and Enterococcus faecium, removal of 97 ± 4%, 97 ± 6% and 86.9 ± 0.8% of paracetamol at 200 mg/L were obtained, respectively, while in the presence of a species belonging to Bacillus cereus group removal of the drug below the limits of detection was attained with evidence of mineralisation, after 144 h of incubation. During the degradation process, the metabolites 4-aminophenol, hydroquinone and 2-hexenoic acid were detected. As far as we know, these species are herein first-time described as paracetamol degraders.FCT: UIDB/04326/2020; European Comission:0483_PROBIOMA_5_Einfo:eu-repo/semantics/publishedVersio

An autochthonous aerobic bacterial community and its cultivable isolates capable of degrading fluoxetine

BACKGROUND Fluoxetine is an antidepressant and recalcitrant fluorine pharmaceutical that is poorly biodegraded, so it enters the hydric resources and causes hazardous effects to aquatic environments. According to these fluoxetine features, the main aim of the present research was to find resistant bacteria in environmental samples with a high degradation efficiency. RESULTS The results obtained from raw municipal wastewater spiked with fluoxetine and inoculated with aerobic sludge from a Portuguese wastewater treatment plant under highly aerobic conditions showed that more than half and approximate to 89% of the drug was degraded after 48 and 144 h, respectively. During the assay, the initial population (mainly composed of Arcobacter, Bacteroides, and Macellibacteroides) shifted with an increase of members of the Acinetobacter, Rheinheimera, Shewanella, Pseudomonas, Methylobacillus, Piscinobacter genera and Aeromonadales order and the Pseudomonadaceae family, all of which were likely responsible for fluoxetine biodegradation. From the same sludge, six bacterial isolates were selected and identified as follows: Pseudomonas putida, Enterobacter ludwigii, Pseudomonas nitritireducens, Alcaligenes faecalis, Pseudomonas aeruginosa, and Pseudomonas nitroreducens; all of them grew with fluoxetine as sole carbon source. Pseudomonas nitroreducens showed the highest removal of 55 +/- 1% at 20 mg L-1 fluoxetine after 24 h. CONCLUSION An autochthonous aerobic bacterial community and its cultivable isolates showed the capacity to biodegrade fluoxetine. Biodegradation, rather than adsorption, appears to play the main role in the fluoxetine removal in aerobic conditions using bacteria simply obtained from an environmental sample. As far as is known, those bacteria are reported for the first time as fluoxetine biodegraders; thus, these bacteria are a promising option to integrate into new bioremediation processes aiming at the removal of fluoxetine.Portuguese Foundation for Science and Technology European Commission: UIDB/04326/2020; European Regional Development Fund: 0483_PROBIOMA_5_Einfo:eu-repo/semantics/publishedVersio

Photodegradation of chloramphenicol and paracetamol using PbS/TiO2 nanocomposites produced by green synthesis

The present study describes the photocatalytic potential of the successfully synthesized nanocrystalline PbS/TiO2 nanocomposites in the photodegradation of chloramphenicol and paracetamol. PbS and PbS/TiO2 nanoparticles were synthesized using biological sulphide produced by sulphate-reducing bacteria in batch and in a coupled bioremediation system (upflow anaerobic packed-bed reactor) for acid mine drainage treatment, yielding near-complete metal precipitation (~ 100–99%, respectively). The PbS and PbS/TiO2 composites obtained using sulphide generated in batch have an average particle size ranging from 17 to 25 nm and 15 to 20 nm, respectively, while in bioreactor, both PbS and PbS/TiO2 particles have a similar size range from 20 to 50 nm. All the produced particles presented crystalline cubic structure. The specific surface area of TiO2 and PbS/TiO2 was estimated to be 46.559 m2/g and 38.005 m2/g, respectively. Chloramphenicol removal by photolysis was about 61% after 60 min of Hg irradiation and 36% under sunlight exposition. Chloramphenicol photodegradation using PbS/TiO2 as catalyst was successfully performed in a photoreactor (Hg medium pressure, 450 W) and under solar exposition with a high drug removal efficiency of 96% and 93% after 60 min and 240 min irradiation, respectively. Using TiO2 as a catalyst for photodegradation achieved 98% removal for both Hg and sunlight irradiation (UV index ranging 7–8) after 60 min and 240 min, respectively. Paracetamol removal by photolysis was about 18%. Drug’s photocatalytic degradation using PbS/TiO2 was successfully performed under sunlight exposition with a high removal efficiency of 93%, while in the presence of TiO2, the removal was complete, after 235 min irradiation.UIDB/04326/2020, UID/QUI/00100/2019info:eu-repo/semantics/publishedVersio