An integrated approach to evaluate emerging contaminants in drinking water

The chemical stock of emerging contaminants in Brazilian drinking water is of great interest due to the poor water quality at surface water intakes. In addition, little is known about the effect of some contaminants, such as endocrine disrupting chemicals (EDCs), which may be present in both raw and treated water. The aim of this work was to evaluate selected emerging contaminants in Brazilian waters using both chemical and biological analyses. Sampling sites were established in different municipalities based upon raw water quality data. Estrone, 17 beta-estradiol, estriol, 17 alpha-ethinylestradiol, bisphenol A, 4-n-octylphenol and 4-n-nonylphenol were determined in the samples by liquid chromatography-tandem mass spectrometry. A yeast assay using a Saccharomyces cerevisiae bioluminescent bioreporter was used to evaluate the estrogenic activity of the water samples. The first integrated results revealed similarities between the two individual approaches, since higher values for the bioassay were accompanied by significant concentrations of some selected compounds in surface water samples. No estrogenicity was observed for drinking water samples. Our results also indicate that the usual paradigm of evaluating water quality by measuring selected EDCs in a given water sample via chemical analysis, needs to be reviewed since the observed estrogenicity of a water sample is now a better guiding parameter to the selection of samples and substances to be chemically investigated in further analysis. So far, the data bank produced in this work, i.e., the comparison between chemical burden and observed estrogenicity, is not yet sufficiently robust to fully guide this decision. (C) 2011 Elsevier B.V. All rights reserved.

CHEMICAL CHANGES AND ZINC PHYTOAVAILABILITY IN SEWAGE SLUDGE-AMENDED SOIL ESTIMATED BY THE ISOTOPIC METHOD

CHEMICAL CHANGES AND ZINC PHYTOAVAILABILITY IN SEWAGE SLUDGE-AMENDED SOIL ESTIMATED BY THE ISOTOPIC METHOD. Zn availability in Red Latossol (Rhodic Ferralsol) of different pH amended with different rates of sewage sludge was studied by the isotopic Zn-65 L value method. Soil chemical properties were found to be altered by SS addition. Zn concentration and Zn derived from SS (ZnpfSS) in plant, and Zn phytoavailability (L value), were increased with increasing SS rates. The linear correlation coefficient of plant Zn with SS rates and with L value was significant at 1% probability. The L value proved an efficient method for predicting Zn phytoavailability in sewage sludge-amended soil with different pH under the soil conditions studied.

Sewage sludge does not induce genotoxicity and carcinogenesis

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3rd week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P+ AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group.

NUTRITIONAL BALANCE, ESSENTIAL OIL AND WOOD PRODUCTION FROM Corymbia citriodora Hill & Johnson WITH SEWAGE SLUDGE AND MINERAL FERTILIZER APPLICATIONS

The Corymbia citriodora is one of the most important forest species in Brazil and the reason is the diversity of its use, because it produces good quality wood and the leaves may be used for essential oil production. Although, there are not many studies about species and the handling effect in the nutritional balance. This study aimed to evaluate the biomass production and nutrient balance in the conventional production of essential oil and wood of Corymbia citriodora with sewage sludge application. The experiment design established was the randomized blocks, with four replicates and two treatments: 1 - fertilization with 10 tons ha(-1) (dry mass) of sewage sludge, supplemented with K and B, and 2 - mineral fertilization. It was evaluated the aerial biomass production, the nutrient export of the leaves, the essential oil and wood production at four years old. The trees that received application of sewage sludge produced 20 % more leaves biomass than the trees with mineral fertilization, resulting in larger oil production. Besides, the trees with sewage sludge application produced 14.2 tons ha(-1) yr(-1) of woody biomass that was 27 % higher than the treatment with mineral fertilization. For both treatments the N balance was negative, but treatment with sewage sludge application (-45 kg ha(-1)) was four times lower than the observed on mineral fertilization treatment (-185 kg ha(-1)). It may be concluded in this paper that the application of sewage sludge benefits the production of leaves biomass, essential oil and wood, besides result better nutritional balance of the Corymbia citriodora production system.

A novel xylan degrading beta-D-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular beta-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. beta-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 A degrees C and 3.0-5.5, respectively. beta-xylosidase was stable in acidic pH (3.0-6.0) and 70 A degrees C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The beta-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-beta-d-xylopyranoside, exhibiting apparent K-m and V-max values of 0.66 mM and 39 U (mg protein)(-1) respectively, and to a lesser extent p-nitrophenyl-beta-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel beta-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by beta-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

Treatment of Domestic Sewage in an Anaerobic-Aerobic Fixed-bed Reactor with Recirculation of the Liquid Phase

This study reports the performance of a combined anaerobic-aerobic packed-bed reactor that can be used to treat domestic sewage. Initially, a bench-scale reactor was operated in three experimental phases. In the first phase, the anaerobic reactor was operated with an average organic matter removal efficiency of 77% for a hydraulic retention time (HRT) of 10 h. In the second phase, the reactor was operated with an anaerobic stage followed by an aerobic zone, resulting in a mean value of 91% efficiency. In the third and final phase, the anaerobic-aerobic reactor was operated with recirculation of the effluent of the reactor through the anaerobic zone. The system yielded mean total nitrogen removal percentages of 65 and 75% for recycle ratios (r) of 0.5 and 1.5, respectively, and the chemical oxygen demand (COD) removal efficiencies were higher than 90%. When the pilot-scale reactor was operated with an HRT of 12 h and r values of 1.5 and 3.0, its performance was similar to that observed in the bench-scale unit (92% COD removal for r = 3.0). However, the nitrogen removal was lower (55% N removal for r = 3.0) due to problems with the hydrodynamics in the aerobic zone. The anaerobic-aerobic fixed-bed reactor with recirculation of the liquid phase allows for concomitant carbon and nitrogen removal without adding an exogenous source of electron donors and without requiring any additional alkalinity supplementation.

Growth and nitrogen fixation in soybean treated with doses of composted sewage sludge

The use of sewage sludge is a highly promising practice for the development of sustainable agricultural systems. The objective of this study was to evaluate doses of sewage sludge composted with and without Rhizobium inoculation in leaf N content, nodule number, nodule dry weight and plant during flowering. The experiment was conducted in the greenhouse of the Department of Soil Science and Natural Resources College of Agricultural Sciences of Botucatu, using as substrate used in vessels of 30 liters a Red Yelow Latosol sandy texture with experimental design adopted was randomized blocks constituted for 10 treatments and five doses of composted sewage sludge (0, 10, 20, 30, 40 t ha(-1)) with or without inoculation Bradyrhizobium japonic with three replications. There was an increase in the number and dry weight of nodules and shoot dry mass of soybeans due to the increase of the dose of sludge up to a dose of 20 t ha(-1) and after this dose there was a decrease of these parameters. At a dose of 10 t ha(-1) sludge compost inoculated seeds showed higher for foliar concentrations of N and number of nodules compared with uninoculated seeds. At a dose of 30 t ha(-1) inoculated seeds were higher compared to uninoculated in all parameters.

Purification of bromelain from pineapple wastes by ethanol precipitation

Bromelain is an aqueous extract of pineapple that contains a complex mixture of proteases and non-protease components. These enzymes perform an important role in proteolytic modulation of the cellular matrix in numerous physiologic processes, including anti-inflammatory, anti-thrombotic and fibrinolytic functions. Due to the scale of global production of pineapple (Ananas comosus L.), and the high percentage of waste generated in their cultivation and processing, several studies have been conducted on the recovery of bromelain. The aim of this study was to purify bromelain from pineapple wastes using an easy-to-scale-up process of precipitation by ethanol. The results showed that bromelain was recovered by using ethanol at concentrations of 30% and 70%, in which a purification factor of 2.28 fold was achieved, and yielded more than 98% of the total enzymatic activity. This enzyme proved to be susceptible to denaturation after the lyophilization process. However, by using 10% (w/v) glucose as a cryoprotector, it was possible to preserve 90% of the original enzymatic activity. The efficiency of the purification process was confirmed by SDS-PAGE, and native-PAGE electrophoresis, fluorimetry, circular dichroism and FTIR analyzes, showing that this method could be used to obtain highly purified and structurally stable bromelain. (C) 2012 Elsevier B.V. All rights reserved.

Soil properties in a sugarcane plantation after the application of treated sewage effluent and phosphogypsum in Brazil

In a field experiment performed in Lins County (Sao Paulo State, Brazil), treated sewage effluent (TSE) irrigation increased sugarcane yield but caused an excessive increase in the exchangeable sodium percentage (ESP) and clay dispersion after 16 months due to an intense irrigation regime (2500 mm/16 months) with sodium rich effluents. After two additional complete cycles with lower TSE irrigation rates (1200 mm year(-1)), 1700 kg ha(-1) of phosphogypsum was added to a section of the irrigated plots to evaluate its residence time and its implications on Na+ dynamics and other soil properties. Undisturbed soil cores were taken 2 years after phosphogypsum application to verify soil physical properties up to 0.2 m depth, and disturbed soil samples were taken every year up to 1 m depth for chemical analyses. After 5 years of consecutive TSE irrigation (2005-2010), soil acidity (pH approximate to 5) and basic cations (Ca approximate to 12, Mg approximate to 6 and K approximate to 2 mmol(c) kg(-1)) were maintained in adequate conditions for plant development without the necessity of liming, while acidity was increased (pH approximate to 4.5) and Ca (approximate to 9 mmol(c) kg(-1)), and the Mg (approximate to 4.5 mmol(c) kg(-1)) concentration decreased in the rainfed without phosphogypsum treatment. An increase in water retention capacity at -30 (from 0.14 to 0.17 m(3) m(-3)) and -1500 kPa (from 0.08 to 0.12 m(3) m(-3)) potentials was also observed in all TSE irrigated treatments. The plots with a phosphogypsum treatment showed average increases of 2 mmol(c) kg(-1) of Ca2+ and 7 mg kg(-1) of S-SO42- in all soil profiles and an average reduction of 2 mmol(c) kg(-1) of Na+ up to 0.4 m from 2008 to 2009. However, the extent of the chemical effects was greater after the first year compared to the second year. The high concentration of Na+ found in previous studies performed in the same area returned to low concentrations after continued TSE irrigation at lower rates, even without the phosphogypsum application. An unusual phosphorus migration was observed to the 0.4-0.8 m soil layer as a result of TSE irrigation, most likely due to a high pH and a Na carbonate-dominated TSE. (C) 2012 Elsevier B.V. All rights reserved.

A comparative study of the purification of betanin

Betanin is a natural pigment with antioxidant properties used as a food colourant. This work describes the spectrophotometric and chromatographic quantification of betanin (2S/15S) and its epimer isobetanin (2S/15R) in fresh beetroot juice, food-grade beetroot powder and betanin standard diluted in dextrin. Absorption spectra of all three samples were deconvoluted using a mixed three-function model. Food-grade beetroot powder has the largest amount of violet-red impurities, probably formed during processing. The purification of betanin from these complex matrices was carried out by seven different methods. Ion exchange chromatography was the most efficient method for the purification of betanin from all samples; however, fractions contain high amounts of salt. Reversed-phase HPLC as well as reversed-phase column chromatography also produced good results at a much faster rate. The longer retention time of isobetanin when compared to betanin in reversed-phase conditions has been investigated by means of quantum-mechanical methods. (C) 2011 Elsevier Ltd. All rights reserved.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

Abstract Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments.

Bioassay guided purification of the antimicrobial fraction of a Brazilian propolis from Bahia state

Abstract Background Brazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6. Methods Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity. Conclusion a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.

Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135

Abstract Background The gene coding for the uncharacterized protein PAB1135 in the archaeon Pyrococcus abyssi is in the same operon as the ribonuclease P (RNase P) subunit Rpp30. Findings Here we report the expression, purification and structural analysis of PAB1135. We analyzed the interaction of PAB1135 with RNA and show that it binds efficiently double-stranded RNAs in a non-sequence specific manner. We also performed molecular modeling of the PAB1135 structure using the crystal structure of the protein Af2318 from Archaeoglobus fulgidus (2OGK) as the template. Conclusions Comparison of this model has lead to the identification of a region in PAB1135 that could be involved in recognizing double-stranded RNA.