Continuous removal of caffeine in a horizontal-flow anaerobic immobilized biomass bioreactor: identification of transformation products.

Anaerobic bioreactors are an efficient technology for the biodegradation of emerging contaminants in environmental matrices. In this work, a horizontal-flow anaerobic immobilized biomass (HAIB) bioreactor was used to remove caffeine (CAF), which is frequently found in various aqueous matrices. The acrylic bench top bioreactor, with dimensions of 100 × 5.00 cm, was operated with a hydraulic retention time (HRT) of 12 h, during 45 weeks, under mesophilic conditions. The operation was performed in 4 phases: without CAF addition (phase I); CAF spiked at 300 µg L-1 (phase II); CAF at 600 µg L-1 (phase III); and CAF at 900 µg L-1 (phase IV). Samples of bioreactor influent and effluent were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The bioreactor removed organic matter (OM) and CAF with efficiencies of 88 and 93%, respectively. The first-order apparent removal constant (Kapp) values for OM and CAF were 0.419 and 0.304 h-1, respectively. Five transformation products (TPs) were identified, with m/z 243, 227, 211, and 181 (two products). The HAIB bioreactor is a suitable system for the removal of CAF present in wastewater, even at a concentration level of µg L-1.

Automated method to determine pharmaceutical compounds in wastewater using on-line solid-phase extraction coupled to LC-MS/MS.

An automated method was developed using on-line solid-phase extraction (SPE) as a sample preparation step, coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS), for determination of pharmaceutical compounds in wastewater samples at nanograms per liter to micrograms per liter concentration levels. This method is suitable for use in routine analysis, especially in environmental applications, avoiding cross-contamination and requiring minimal sample handling. Results can be obtained rapidly, with a chromatographic run time of only 24 min (including sample preparation and chromatographic analysis). Using a 50 µL injection volume, the method was validated according to international guidelines, considering parameters included in terms of method detection (MDL) and quantification limit (MQL), linearity, inter-day and intra-day precisions, and matrix effects. Assessment of chromatographic efficiency considered peak resolution and asymmetry, and carryover was evaluated to ensure analytical reliability and the ability to reuse the SPE cartridge. The intra- and inter-day precisions were lower than 10 and 17%, respectively. The MDL values ranged from 1×10-6 to 1 µg L-1, while the MQL values were from 0.001 to 3 µg L-1. Matrix effects were minimized by isotope dilution calibration. Application of the method to 20 wastewater samples showed that caffeine was the most frequently detected compound, with the highest concentration of 715 µg L-1, while other pharmaceutical compounds were detected in fewer samples and at lower concentrations (up to 8.51 µg L-1).

Identification of organic contaminants in vinasse and in soil and groundwater from fertigated sugarcane crop areas using target and suspect screening strategies.

This work evaluated for the first time the sustainability of vinasse reuse as a fertilizer in sugarcane crops by assessing the occurrence of organic contaminants and their potential for dissemination to soils and groundwater in fertigated areas. A comprehensive screening of organic contaminants was performed in vinasse, soil and groundwater using target analysis, to investigate the occurrence of multiple-class antibiotics, in combination with suspect screening using NORMAN Digital Sample Freezing Platform. Even though antibiotics are used in the ethanol production process and were expected to be ubiquitous contaminants, they were not detected in any of the samples. Nevertheless, the HRMS-based wide-scope suspect screening (including >7800 substances such as pharmaceuticals, agrochemicals, preservatives and industrial chemicals) allowed the tentative identification of 56 compounds, mostly pesticides, food additives, industrial and naturally occurring substances. Results showed no overlap between the compounds detected in vinasse and environmental samples, suggesting that the pollutants found in soil and groundwater might come from alternative sources other than vinasse reuse.

Pressurized Liquid Extraction (PLE) and QuEChERS evaluation for the analysis of antibiotics in agricultural soils.

Vinasse, a liquid waste which originates from the production of ethanol fuel from sugarcane, has been widely used as soil amendment in Brazil. An important concern that arises from vinasse reuse is the dissemination of antibiotics to the environment through crop soils. This work evaluated the performance of Pressurized Liquid Extraction (PLE) and QuEChERS (quick, easy, cheap, effective, rugged and safe) to extract several multiple-class antibiotics, such as cephalosporins, fluoroquinolones, ionophores, lincosamides, macrolides, quinolones, streptogramin, sulfonamides, tetracyclines and others, from agricultural soils. The performance of several parameters was evaluated for both PLE and QuEChERS, such as the extraction temperature (for PLE), solvents composition, pH and the addition of EDTA. Both methods were able to extract most target antibiotics. However, QuEChERS showed higher recoveries for macrolides and nitroimidazoles, while PLE was more suitable for fluoroquinolones and ionophores (i.e. monensin). The use of citrate-phosphate buffer at pH 7.0, in combination with methanol for PLE and with acetonitrile for QuEChERS, provided the highest antibiotic recoveries for both methods. The use of EDTA did not increase antibiotic recovery rates for QuEChERS, while the temperature had almost no influence on the extraction efficiency in PLE.•Citrate-phosphate buffer at pH 7.0 provided higher antibiotic recoveries for QuEChERS and PLE.•The combination buffer-methanol provided higher recoveries for PLE.•QuEChERS and PLE methods were able to extract most of the target antibiotics.

Comparison of Two Methods for Counting Molds in Fermentations Using the Production of Bikaverin by Fusarium oxysporum CCT7620 as a Model.

The dry cell weight (DCW) measurement is one of the preferred methods to determine the growth of filamentous fungi. However, this technique is not applicable to insoluble culture media, besides being possibly influenced by the presence of extracellular biomass. The standard plate counting (SPC) is a reference method for detecting viable cells; however, it is referred as imprecise. In this study, we did a comprehensive analysis of the errors associated to each procedure and also determined the growth kinetics of Fusarium oxysporum in soluble (DCW and SPC) and insoluble (SPC) culture media. Finally, we used the production of bikaverin in airlift bioreactor containing insoluble medium as a case study to estimate red pigment production and to monitor biomass growth via SPC. We concluded that SPC can be used to give reliable fungal growth kinetics in media with insoluble matter, yielding errors equivalent to DCW depending on the number of replicates done for serial dilutions and plate counting.

Occurrence of PPCPs in a Brazilian water reservoir and their removal efficiency by ecological filtration.

The presence of PPCPs (Pharmaceuticals and Personal Care Products) in water sources and drinking water has concerned researchers in recent times. This study was carried out to evaluate the occurrence of 6 PPCPs (namely paracetamol, diclofenac, naproxen, ibuprofen, benzophenone-3 and methylparaben) in the Lobo reservoir, their degradation products, and how efficiently they were removed by 22 ecological filters, considering individual and mixture of compounds. There were 3 spiking events of PPCPs (2 µg L-1) in the ecological filter influents conducted with a lag period of 15 days between spikes. Water samples were collected from the influent and effluent of the filters at 3, 6 and 24 h after each spiking event. All target PPCPs were identified in the Lobo reservoir water in the range of µg L-1. The personal care products were detected with 100% frequency in the samples, and in higher concentrations compared to the pharmaceuticals. Degradation products of diclofenac and benzophenone-3 were identified in the water samples. Results of this investigation show that ecological filtration was an effective process (70-99%) to remove 2 µg L-1 of the selected PPCPs, and demonstrated that the filters were resilient to individual compounds and to their mixtures.

Quantifying the contribution of dyes to the mutagenicity of waters under the influence of textile activities.

The combination of chemical analyses and bioassays allows the identification of potentially mutagenic compounds in different types of samples. Dyes can be considered as emergent contaminants and were detected in waters, under the influence of textile activities. The objective of this study was to evaluate the contribution of 9 azo dyes to the mutagenicity of representative environmental samples. Samples were collected along one year in the largest conglomerate of textile industries of Brazil. We analyzed water samples from an important water body, Piracicaba River, upstream and downstream two main discharges, the effluent of a wastewater treatment plant (WWTP) and the tributary Quilombo River, which receives untreated effluent from local industries. Samples were analyzed using a LC-MS/MS and tested for mutagenicity in the Salmonella/microsome microsuspension assay with TA98 and YG1041. Six dyes were detected in the collected samples, Disperse Blue 291, Disperse Blue 373, Disperse Orange 30, Disperse Red 1, Disperse Violet 93, and Disperse Yellow 3. The most sensitive condition for the detection of the mutagenicity was the strain YG1041 with S9. The concentration of dyes and mutagenicity levels varied along time and the dry season represented the worst condition. Disperse Blue 373 and Disperse Violet 93 were the major contributors to the mutagenicity. We conclude that dyes are contributing for the mutagenicity of Piracicaba River water; and both discharges, WWTP effluent and Quilombo River, increase the mutagenicity of Piracicaba River waters in about 10-fold. The combination of chemical analysis and bioassays were key in the identification the main drivers of the water mutagenicity and allows the selection of priority compounds to be included in monitoring programs as well for the enforcing actions required to protect the water quality for multiple uses.

Design, synthesis and characterization of a hexapeptide bio-inspired by acetylcholinesterase and its interaction with pesticide dichlorvos.

This paper describes the molecular modeling design, synthesis and characterization of a new bio-inspired hexapeptide of acetylcholinesterase enzyme and its interaction with the organophosphate pesticide dichlorvos monitored by UV-Vis spectroscopy and mass spectrometry. This strategy can contribute to the development of synthetic receptors to be coupled to biosensor transducers, avoiding the issues associated with proteins such as low stability under different pH and temperature conditions and high production cost. The resulting data of this work indicate a strong interaction between the pesticide dichlorvos and the hexapeptide (NH3(+)-Glu-His-Gly-Gly-Pro-Ser-COO(-)) with a binding constant of 4.10 × 10(5) M(-1) and the formation of an adduct by covalent binding on the serine residue from the hexapeptide.

Diclavatol and tetronic acids from Penicillium griseoroseum.

In our continuous studies on the chemistry of the endophytic fungus Penicillium griseoroseum, an endophyte isolated from fruits of Coffea arabica, we isolated clavatol, a dimethylated tetraketide, and its dimer which appears to be a novel natural compound. The studies also resulted in the identification of two known tetronic acids, viridicatic acid and terrestric acid, found in ethyl acetate and n-butanol extracts. Spectroscopic studies using 1-D and 2-D NMR and MS/MS analysis were performed to determine the structures of these compounds, first reported by this Penicillium. Two other tetronic acids congeners were identified through HPLC/MS/MS studies, based on fragmentation pattern of ions produced from ionised tetronic acids, and UV light absorptions.

Oxidative potential of some endophytic fungi using 1-indanone as a substrate.

The oxidative potential of the fungus Penicillium brasilianum, a strain isolated as an endophyte from a Meliaceae plant (Melia azedarach), was investigated using 1-indanone as a substrate to track the production of monooxygenases. The fungus produced the dihydrocoumarin from 1-indanone with the classical Baeyer-Villiger reaction regiochemistry, and (-)-(R)-3-hydroxy-1-indanone with 78% ee. Minor compounds resulting from lipase and SAM activities were also detected. The biotransformation procedures were also applied to a collection of Penicillium and Aspergillus fungi obtained from M. azedarach and Murraya paniculata. The results showed that Baeyer-Villiger were mostly active in fungi isolated from M. azedarach. Almost all of the fungi tested produced 3-hydroxy-1-indanone..

Biosynthesis of phenylpropanoid amides by an endophytic Penicillium brasilianum found in root bark of Melia azedarach.

Biosynthetic studies on brasiliamides, potently convulsive and bacteriostatic compounds from an endophytic Penicillium brasilianum isolated from Melia azedarach (Meliaceae), confirms their phenylpropanoid origin, which is very uncommon in fungi. Feeding experiments with [2-(13)C]- phenylalanine indicated the incorporation of two units of this amino acid on brasiliamide structures. The first step in the phenylpropanoid pathway to those compounds was evaluated through enzymatic bioassays and confirmed the phenylalanine ammonia-lyase (PAL) participation. The metabolism of phenylalanine in this fungus is discussed.