Robson José de Cássia Franco Afonso

Photolysis and photocatalysis of ibuprofen in aqueous medium: characterization of by-products via liquid chromatography coupled to high-resolution mass spectrometry and assessment of their toxicities against Artemia Salina

The degradation of the pharmaceutical compound ibuprofen (IBP) in aqueous solution induced by direct photolysis (UV-A and UV-C radiation) and photocatalysis (TiO2 /UV-A and TiO2 /UV-C systems) was evaluated. Initially, we observed that whereas photocatalysis (both systems) and direct photolysis with UV-C radiation were able to cause an almost complete removal of IBP, the mineralization rates achieved for all the photodegradation processes were much smaller (the highest value being obtained for the TiO2 /UV-C system: 37.7%), even after an exposure time as long as 120 min. Chemical structures for the by-products formed under these oxidative conditions (11 of them were detected) were proposed based on the data from liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) analyses. Taking into account these results, an unprecedented route for the photodegradation of IBP could thus be proposed. Moreover, a fortunate result was achieved herein: tests against Artemia salina showed that the degradation products had no higher ecotoxicities than IBP, which possibly indicates that the photocatalytic (TiO2 /UV-A and TiO2 /UV-C systems) and photolytic (UV-C radiation) processes can be conveniently employed to deplete IBP in aqueous media.

Occurrence of endocrine disrupting compounds in water sources of Belo Horizonte Metropolitan Area, Brazil.

Endocrine disrupting compounds (EDCs) have attracted the attention of the scientific community because of the effects of EDCs on aquatic fauna and the potential threat they pose to human health. There are a handful of papers on the monitoring of EDCs in Brazilian surface waters, hence this research was aimed at assessing, by using liquid chromatography coupled to mass spectrometry (LC–MS), the presence of 17β‐estradiol, 17α‐ethynylestradiol and 4‐nonylphenol in surface waters used for supplying the Belo Horizonte Metropolitan Area, Minas Gerais state, Brazil. The one‐year monitoring period of three water sources showed that 4‐nonylphenol was detected in all samples in a concentration range of 44 to 1918 ng L−1, whilst the natural and synthetic estradiols were hardly detected (only in ∼15% of samples) and always in low concentrations (2 to 54 ng L−1). Samples of partially treated water, collected in three water treatment plants before the chlorination step, showed that the steps of prechlorination, flocculation–sedimentation and sand filtration did not efficiently remove the EDCs studied.

Determination of endocrine‐disrupting compounds in waters from Rio das Velhas, Brazil, by liquid chromatography/high resolution mass spectrometry (ESI‐LC‐IT‐TOF/MS)

Some organic microcontaminants, known as endocrine‐disrupting compounds (EDC), are frequently found in surface waters and are of concern because of their ability to alter the normal functioning of the endocrine system. In this study five of these EDCs (estradiol, ethynylestradiol, bisphenol A, nonylphenol and diethylphthalate) in surface water samples, collected at the upper part of Rio das Velhas river (located between Ouro Preto and Nova Lima, Minas Gerais state, Brazil) were analysed. The analytical methodology, which employed solid‐phase extraction and high‐performance liquid chromatography coupled to high resolution mass spectrometry (HPLC‐MS/MS), was properly validated and resulted in limits of quantification (LOQ, S/N = 10) of 4.3 ng/L for diethylphtalate, 3.9 ng/L for nonylphenol, 3.6 ng/L for estradiol, 4.7 ng/L for ethynylestradiol and 2.5 ng/L for bisphenol. The five compounds were monitored for eight months, from sites where the hormones were occasionally found. Ethynylestradiol and estradiol concentrations varied from 5.6 to 63.8 ng/L. Bisphenol was present in all samples in a concentration that ranged from 8.6 to 168.3 ng/L. The other two compounds were at quantifiable levels in all collected samples, with nonylphenol varying from 25.9 to 1435.3 ng/L and diethylphthalate from 5.0 to 410.9 ng/L. The study showed that nonylphenol pollution seemed to originate from agricultural run‐off, while diethylphthalate pollution originated from the discharge of domestic sewage. Risk analysis studies showed that the environmental concentrations of nonylphenol and diethylphthalate do not present risks to human health; however, the concentrations found for bisphenol and ethinylestradiol could be toxic to humans, especially infants, if such compounds are not effectively removed at water treatment plants.

Determinação de carbamato de etila em aguardentes de cana por CG-EM.

A simple, specific and sensitive GC-MS procedure with ion m/z 62 was validated for the determination of ethyl carbamate (EC) in spirits. It exhibited linearity over the concentration of 30 to 600 μg/L with 30 μg/L limit of quantification. EC was detected in 70 of the 71 samples analyzed with levels from 33 to 2609 μg/L (mean level = 893 μg/L). 35% of the samples contained 500 to 1000 μg/L and 23% contained 150 to 500 and 1000 to 1500 μg/L. No significant correlation was found between EC and the levels of copper, pH and alcohol content of the samples.

Behaviour of pharmaceuticals and endocrine disrupting chemicals in simplified sewage treatment systems.

This work assessed the behaviour of nine pharmaceuticals and/or endocrine disrupting chemicals (EDCs) in demo-scale upflow anaerobic sludge blanket reactors (UASB reactors) coupled to distinct simplified post-treatment units (submerged bed, polishing ponds, and trickling filters) fed on raw sewage taken from a municipality in Brazil. The dissolved concentration of the studied micropollutants in the raw and treated sewage was obtained using solid phase extraction (SPE) followed by analysis in a liquid chromatography system coupled to a hybrid high resolution mass spectrometer consisting of an ion-trap and time of flight (LC-MS-IT-TOF). The UASB reactors demonstrated that they were not appropriate for efficiently removing the assessed compounds from the sewage. Furthermore, this study demonstrated that the hydraulic retention time (HRT) was an important parameter for the removal of the hydrophilic and less biodegradable compounds, such as trimethoprim and sulfamethoxazole. The post-treatment units substantially increased the removal of most target micropollutants present in the anaerobic effluents, with a greater removal of micropollutants in simplified systems that require a large construction area, such as the submerged bed and polishing ponds, probably because of the higher HRT employed. Alternatively, compact post-treatment systems, such as trickling filters, tended to be less effective at removing most of the micropollutants studied, and the type of packing proved to be crucial for determining the fate of such compounds using trickling filters.

Soluble microbial product (SMP) characterization in bench-scale aerobic and anaerobic CSTRS under different operational conditions.

Abstract - This work presents results on the production and characterization (by both mass spectrometry and conventional chemical analyses) of Soluble Microbial Products (SMP) that accumulated in aerobic and anaerobic bench scale completely stirred tank reactors (CSTRs) fed with glucose or acetate under different hydraulic retention times (HRT) and temperatures. SMP accumulation varied from 2 to 68% of the influent COD in the aerobic reactor and from 9 to 27% in the anaerobic reactor and increased with the decrease in temperature and with the HRT reduction in the aerobic reactor. On the other hand, in the anaerobic reactor, the organic loading rate and the temperature had little impact on SMP production, implying that the SMP originated from different mechanisms in each system. For both reactors, a higher accumulation of SMPs was observed as the substrate was acetate when compared to glucose, and the chemical analysis showed that the majority of the SMP did not seem to be proteins or carbohydrates. Principal component analysis (PCA) of the mass spectra from positive and negative mode electron-spray ionization (LC-IT-TOF-MS) and results from matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the chemical analyses and showed the absence of proteins in the effluents and the predominance of low molecular weight SMP. The PCA analysis also showed that the majority of the SMP from aerobic and anaerobic reactors did not seem to originate from soluble extracellular polymeric substances (EPS) or cell lysis products.

Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5′,5″-trifluor-d,l-leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. “Cachaça” produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in “cachaça” production.

Photodegradation of bisphenol A in aqueous medium: monitoring and identification of by-products by liquid chromatography coupled to high-resolution mass spectrometry.

RATIONALE Environmental bisphenol A (BPA) contamination is currently a matter of concern. This compound can disrupt the endocrine system by mimicking natural hormones and cause adverse effects on different organisms. In addition, it has been suggested that BPA can impair brain development, especially in fetuses and children. To efficiently remove BPA from contaminated water and wastewaters, several emerging technologies have been developed. Most are based on photodegradation. However, by-products resulting from the application of such methods have not been properly characterized. METHODS The photodegradation of BPA was conducted using two different methods: photocatalysis (TiO2/UV-A and TiO2/UV-C systems) and direct photolysis (UV-A and UV-C radiation). The degradation process was continuously monitored to identify and observe the by-products formed under these conditions. Direct infusion electrospray ionization coupled to high-resolution mass spectrometry in negative ion mode [ESI(-)-HRMS] and liquid chromatography coupled to high-resolution mass spectrometry (HPLC/HRMS) were employed to monitor these by-products. RESULTS The results revealed that the TiO2/UV-C system was the most efficient in causing both BPA depletion and mineralization of the organic matter in solution. Moreover, accurate mass data allowed for the assignment of molecular formulae for seven by-products, many of them unprecedented, formed under these conditions. Thus, a route for the photodegradation of BPA in aqueous medium could be proposed based on these results. Finally, additional tests revealed that the by-products showed a higher toxicity against Artemia salina than the primary precursor. CONCLUSIONS All of these findings indicate that the identification of by-products arising from oxidative treatments is of primary importance because such compounds can be more hazardous than their precursors.

Biotechnological potential of yeast isolates from cachaça: the Brazilian spirit

This study identified phenotypic traits appropriate for biotechnological applications of 118 yeasts isolated from cachaça distilleries. Different properties were verified: capacity to use alternative carbon sources; ability to tolerate high concentrations of sucrose, ethanol, methanol, aluminum and zinc as well as different pH values and foam production. Pichia guilliermondii and Pichiaanomala strains were identified as the most promising ones for application in the second-generation biofuel industry, showing ability to grow on high glycerol concentrations. Other isolates, identified as Saccharomyces cerevisiae, produced bioethanol comparable to the industrial strains, and were therefore ideal for use in the first-generation ethanol industry. Some of these strains also showed high resistance to aluminum, as observed in sugarcane juice, and to inter-cycle washings with diluted sulphuric acid, as performed in the industrial bioethanol production process. In summary, yeast isolates from cachaça distilleries displayed robustness and phenotypic plasticity, which makes them interesting for biotechnological applications.

Molecular characterization of SUMO E2 conjugation enzyme: differential expression profile in Schistosoma mansoni

SUMO-dependent post-translational modification is implicated in a variety of cellular functions including gene expression regulation, nuclear sub-localization, and signal transduction. Conjugation of SUMO to other proteins occurs in a similar process to ubiquitination, which involves three classes of enzymes: an E1 activating, an E2 conjugating, and an E3 target-specific ligase. Ubc9 is the unique SUMO E2 enzyme known to conjugate SUMO to target substrates. Here, we present the molecular characterization of this enzyme and demonstrate its expression profile during the S. mansoni life cycle. We have used bioinformatic approaches to identify the SUMO-conjugating enzyme, the SmUbc9-like protein, in the Schistosoma mansoni databases. Quantitative RT-PCR was employed to measure the transcript levels of SUMO E2 in cercariae, adult worms, and in vitro cultivated schistosomula. Furthermore, recombinant SmUbc9 was expressed using the Gateway system, and antibodies raised in rats were used to measure SmUbc9 protein levels in S. mansoni stages by Western blotting. Our data revealed upregulation of the SmUbc9 transcript in early schistosomula followed by a marked differential gene expression in the other analyzed stages. The protein levels were maintained fairly constant suggesting a post-transcriptional regulation of the SmUbc9 mRNA. Our results show for the first time that S. mansoni employs a functional SUMO E2 enzyme, for the conjugation of the SUMO proteins to its target substrates.