Ultratrace-level determination of N-Nitrosodimethylamine,N-Nitrosodiethylamine,and N-Nitrosomorpholine in waters by solid-phase extraction followed by liquid chromatography-tandem mass spectrometry

N-nitrosamines are a new class of emerging nitrogenous drinking water disinfection by-products. These compounds are probably carcinogenic which could seriously affect the safety of drinking water consumers. The aim of this study is to develop a simple, fast, and specific analytical method for the routine determination of low part per trillion levels of N-nitrosamines in waters. An ultra high pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) method was developed for the qualitative and quantitative analysis of N-nitrosamines in waters. N-nitrosamines were extracted, purified and concentrated from water samples in one step using a solid-phase extraction (SPE). The compounds were detected in multiple reaction monitoring via electrospray ionisation source with positive ionisation mode. To achieve symmetrical peak shapes and a short chromatographic analysis time, the mobile phase consisting of acetonitrile, water and formic acid (60:40:0.1, v/v/v) was used in the experiment. Chromatographic separation of N-nitrosamines was done in less than two minutes. All calibration curves had good linearity (r²≥ 0.9989). The intra- and inter-day precision of the assay ranged from 0.59% to 3.11% and accuracy ranged from 99.66% to 104.1%. The mean recoveries of N-nitrosamines in spiked water were 98%-101%. The reproducability was acceptable with relative standard deviations of less than 3.53%. The proposed method yielded detection limits very low which ranges from 0.04 to 0.16 ng L^?1. Finally, the developed analytical method was successfully applied to the analysis of N-nitrosamines in natural water sample     

Glass foam from window panes and bottle glass wastes

Glass foams are building materials that now compete with classic insulating polymeric and fiber materials for thermal enveloping. The low flammability, high chemical durability and thermal stability are distinct advantages over polymeric materials. The present paper proposes the possibility of producing glass foam using two types of recycled glass wastes (window panes and bottle glass) together with plaster wastes from used ceramic casting molds as foaming agent. Optical microscopy, measurements of apparent porosity and density, hydrolytic and chemical stability, as well as thermal conductivity were used in order to characterize the obtained glass foams as insulator materials for the building industry. The apparent porosity of glass foams ranges between 20.19–54.54% when using window glass wastes, and 18.77–51.75% with bottle glass wastes. Thermal conductivity was less than 0.25 W mK-1 for all the studied glasses. The obtained results confirm that there exists an alternative method for producing glass foams, for example, from glass wastes and used ceramic plaster molds, which are utilized as foaming agents with good chemical stability and insulating properties.     

Assessment ofBeauveria bassiana Nov. EO-1 strain,which produces a red pigment for microbial control

A new strain of the fungusBeauveria bassiana Nov. EO-1 (ATCC 74037), which produces a red pigment in solid and liquid culture, has been isolated from an infected whitefly. The red pigment was extracted and has been identified by mass spectrometry as oosporein, a potent dibenzoquinone mycotoxin. In order to assess the potential of this entomogenous fungi for microbial control purposes, a mycelium bead formulation was developed as a source for pathogenic conidial spores and oosporein production. The mycelium bead preparation was found to be a stable fungal carrier. Conidiation and germination studies have revealed the mycelium bead viability is 100% over a 1-yr period when stored at 4°C. Conidial spore production from the mycelium beads has been falling substantially per time from an initial value of 1.5 × 10⁸ spores per bead to 3 × 10⁵ spores per bead after a year storage at 4°C. However, the mycelium bead formulation continues to produce oosporein on agar media, at the same intensity throughout the 1 yr period. In in vitro and in vivo small scale greenhouse experimentsBeauveria bassiana Nov. EO-1 were compared with known entomogenous fungi,Beauveria sp. andPaceilomyces sp. Beauveria bassiana Nov. EO-1 was found to have a high pathogenicity against foliage insect pests (e.g., whiteflies and mealy bugs), and against soil insects (e.g., citrus root weevils). The utilization of a mycelium bead based on this strain,Beauveria bassiana Nov. EO-1, as a source of conidial spores and oosporein may have broad applications for the control of various insect pests.     

Studies on some enzymes of alginic acid biosynthesis in mucoid and nonmucoidAzotobacter chroococcum strains

Measurements of enzymes involved in alginate biosynthesis were straightforward in mucoid (alginate-positive)Azotobacter chroococcum ATCC 4412 crude extracts. At the stationary growth phase, where the production of the exopolysaccharide was greatest, the enzymes phosphomannose isomerase and GDP-mannose pyrophosphorylase increased markedly, whereas phosphomannomutase and GDP-mannose dehydrogenase kept the high activity levels measured in the acceleration growth phase. In nonmucoid (alginatenegative)A. chroococcum andA. vinelandii strains, the activities of phosphomannose isomerase and GDP-mannose pyrophosphorylase were rather low or, in some cases, undetectables. Except inA. chroococcum MCD1, which exhibited a low activity, phosphomanomutase was high in the nonmucoidAzotobacter strains, and GDP-mannose dehydrogenase reached a significant activity level in two out of four nonmucoid strains tested. The results suggest that derepression of phophomannose isomerase and GDP-mannose pyrophosphorylase is asine qua non condition for alginate formation byA. chroococcum.     

Improved procedure for the preparation of DNA restriction fragments suitable for sequencing

A rapid and integrated procedure was developed for the preparation of small DNA restriction fragments ( ≤ 1000 bp) starting from a large cosmid (35,000 bp) containing exogenous DNA. The process is based on restriction enzymatic digestion followed by HPLC separation and fractions collection. All DNA fragments are separated in a single run, detected “on-line” by UV absorption, and straightforward collected with very high recovery. Small fragments can be directly subjected to the sequence procedure, whereas those larger than 1000 bp are redigested with a second enzyme, the fractionated subfragments are separated, ligated to plas-mid vector, and sequenced. A human genomic cosmid of 35,000 bp (26H7) has been chosen as a model.     

Invertebrate organisms as biological indicators of heavy metal pollution

Some species of invertebrate animals are known to be efficient accumulators of trace elements. Generally, metal accumulation by such organisms is based on efficient detoxification mechanisms, such as intracellular compartmentalization, or metal inactivation by binding to metallothioneins. Metal accumulators have often been used as accumulation indicators of environmental metal pollution. This means that, ideally, metal concentrations in the animal’s body reflect quantitatively or semiquantitatively environmental pollution levels. In reality, however, many factors, such as the animal’s weight and age, can disturb such quantitative relationships. These factors have, therefore, to be considered carefully before an invertebrate is utilized as accumulation indicator for metal pollution. Apart from accumulation, many invertebrates exposed to elevated metal concentrations respond to this stress by metal-induced synthesis of metallothioneins. Additionally, metallothionein in metal-loaded organisms can be present in different isoforms that are specifically synthesized in response to different metals. These facts make metallothionein a potential biomarker for metal stress in invertebrates. One possibility may be to assess parameters of metallothionein synthesis at the molecular or biochemical level. Moreover, metallothionein isoform patterns could provide information on different isoforms synthesized in response to different metals or chemicals. In any case, however, care must be taken to consider intrinsic physiological parameters, such as nutritional or developmental factors, which could also interfere with metallothionein synthesis.     

Cloning of a new allelic variant of aSaccharomyces diastaticus glucoamylase gene and its introduction into industrial yeasts

A new allelic variant of theSTA2 gene, designated asSTA2 ^K, coding for a secreted glucoamylase, was cloned. Differences were revealed both in the structural gene and in the promoter region, as compared to otherSTA genes. The most peculiar structural features ofSTA2 ^K are 1. a 1.1-kb natural deletion in its promoter located 189 nucleotides upstream of the translation start codon; and2. an Asn→ Asp single amino acid change within the putative active site of the encoded glucoamylase. Neither the presence of glucose in the medium nor the host cell's mating type constellation affected the expression level ofSTA2 ^K inS. cerevisiae. Self-replicating yeast plasmids containingSTA2 ^K were constructed and used to transform a laboratory yeast strain and various brewing strains. Pilot brewing tests with glucoamylase-secreting transformants of a brewing strain produced superattenuated beers at accelerated fermentation rates.     

Application of a novel 1,1′-dimethylferricinimum dye for the determination of uric acid in urine

Water-soluble 2-hydroxypropyl-β-cyclodextrin (Hp-β-CyD), a cyclic and nonreducing oligosaccharide was used to enclose a hydrophobic guest molecule 1,1′-dimethylferrocene (DMF) to form a water-soluble yellow complex. At high concentrations (300 mM), Hp-β-CyD enclosed up to 100 mM DMF. The yellow complex was electrochemically oxidized (platinum vs Ag/AgCl poised at +450 mV) to form a blue dye, 1,1′-dimethylferricinium (DMF⁺). This is a one-electron transfer process and the ferricinium cation formed exhibited an absorption peak at 650 nm. The concentrated DMF⁺ was stable for at least 4 mo at 4°C and insensitive to a wide pH variation (pH 2–11). Application of the novel DMF⁺ complex as a colorimetric dye for the determination of uric acid in urine was successfully demonstrated. The reaction between the dye and uric acid is almost instantaneous and decrease in absorbance caused by the reduction of 1,1′-dimethylferricinium to 1,1′-dimethylferrocene can be followed at 650 nm. The results obtained agreed well with those of the reference reversed-phase HPLC method.     

Solid-phase thiolsulfinates for the reversible immobilization of thiols

A new method for the reversible immobilization of thiol-containing substances on agarose beads is presented. It is based on the use of thiolsulfinate (disulfide monoxide) as a solid-phase reactive group. The thiolsulfinate groups are introduced by controlled oxidation of thiol agarose. The method comprises two steps: First, mild oxidation of the agarose thiol groups to disulfide structures with potassium ferricyanide. Second, the oxidation of the so-formed agarose disulfide groups to thiolsulfinate groups by use of a stoichiometric amount of the oxidizing agent magnesium monoperoxyphtalate. The solid-phase thiolsulfinate groups react very easily with thiols, which, as a result of the reaction, will be bound to the agarose beads by disulfide bonds. The adsorbent derivative is very suitable for the reversible immobilization of low as well as high-mol-wt thiols as demonstrated with reduced glutathione, penicillamine, mercaptoethanesulfonic acid, thiolated bovine serum albumin,β-galactosidase, and ±₁-antitrypsine. Since treatment of the agarose derivatives with an excess of low-mol-wt thiols (e.g., dithiothreitol) leads to release of the bound molecules and regeneration of the original thiol groups, the reactive thiolsulfinate groups can easily be regenerated by the mentioned two-step procedure. The cycle of oxidation, binding, reduction, and reoxidation can be performed several times while retaining thiol binding capacity.     

Some properties of a thermostable β-xylosidase fromRhodothermus marinus

The extracellular β-xylosidase (EC 3.2.1.37) excreted by the thermophilic eubacteriumRhodothermus marinus when grown on xylan has been investigated. The enzyme has been partially purified by ultrafiltration and gel filtration, and some of its characteristics are presented.Rhodothermus marinus grew on xylan with μmax= 0.4 h^? and the α-xylosidase activity was 50 nkat/mL after 24 h in a batch fermentation. The α-xylosidase activity had a half-life of more than 1 h at 90°C and of 14 h at 85 °C. At 80°C, 80% of the initial activity remained after 24 h. The initial activity increased with increasing temperature, showing maximal activity at 90°C. The β-xylosidase had a pH-optimum of 6 and was stable in the range between pH 5 and 9. At pH 10 and 11, 82 and 66%, respectively, of the initial activity remained after 24 h when incubated at 65°C. The molecular weight was estimated to be 169,000 dalton by gelfiltration.     

Immobilization of Mercuric reductase from aPseudomonas putida strain on different activated carriers

Mercuric reductase was isolated fromPseudomonas putida KT2442::mer-73 and immobilized on Chromatographic carriers activated by various methods. The immobilization methods for covalent coupling were compared with regard to preservation of enzymatic activity and coupling yields. Highest yields were obtained with carriers bearing the most reactive functional groups. Best results were achieved with tresyl chloride-activated carriers. The optimum binding conditions were found at pH 8. Application of the immobilized mercuric reductase for continuous treatment of Hg(II)-containing water was examined in a fixed bed reactor. Space-time yields up to 510 nmol/min-mL were attained. The kinetics of immobilized enzyme systems were not diffusion-controlled.     

Production and use of glucosyltransferases fromLeuconostoc mesenteroides NRRL B-1299 for the synthesis of oligosaccharides containing α-(1→2) linkages

Glucosyltransferase activities, produced by batch culture ofLeuconostoc mesenteroides NRRL B-1299, were recovered both in the culture supernatant (SGT) and associated with the insoluble part of the culture (IGT). A total glucosyltransferase activity of 3.5 U/mL was measured in batch culture. The enzymes from the supernatant were purified 313 times using aqueous two-phase partition between dextran and PEG phases, yielding a preparation with 18.8 U/mg protein. It was shown that both SGT and IGT preparations catalyze acceptor reactions and transfer the glucose unit from sucrose onto maltose to produce glucooligosaccharides. Some of the glucooligosaccharides synthesized (L_n series) contain α-(l→6) osidic linkages and a maltose residue at the reducing end. They were completely hydrolyzed by glucoamy-lase and dextranase. The other glucooligosaccharides synthesized (Bn series) resisted the action of these enzymes. The tetrasaccharide of this series has been characterized by¹³C NMR. Its structure was determined as 2–O–α–D–glucosylpanose. The oligosaccharides synthesized by the maltose acceptor reaction with the SGT and IGT preparations only differed in the relative amounts in which they were produced. The difference may arise from diffusional limitations appearing when the insoluble catalyst is used. Under the assay conditions, the glucanase resistant oligosaccharide yield was 35% with both glucosyltrans-ferase preparations.     

Determination of reaction kinetics of barley straw using thermogravimetric analysis

Alternaria alternata is well known as a producer of tentoxin as well as some other phytotoxic substances. A new method to prepare protoplasts fromAlternaria alternata, suitable for many purposes, was developed. By use of a mixture of lytic enzymes fromHelix pomatia, andTrichoderma harzianum with the commercial preparation “novozym,” it was possible to prepare protoplasts from all stages of fungal development, including the tentoxin production phase. Optimal incubation conditions led to the conversion of 1 g (wet wt) mycelial cells into 2.3–2.5 × 10⁷ protoplasts within 3–6 h. Submerged as well as surface-grown mycelia were suitable. Optimal stabilization of protoplasts was obtained in 0.8M KCI. The protoplasts were used for both mutagenic treatment and physiological studies. UV irradiation of protoplasts resulted in formation of hyperproducing mutants. Protoplasts were able to form tentoxin. The biosynthetic activity of protoplasts from surface-grown mycelium was 40% that of intact mycelia. Although intact submerged myclia did not synthesize tentoxin, protoplasts of both types of mycelia produced this toxin, indeed protoplasts from submerged mycelia were even more active than those from surface mycelia. Neither oxygen tension nor mechanical stress during the shaking culture is the reason for the lack of tentoxin production by intact submerged mycelial pellets. Since tentoxin-synthesizing enzymes were apparently present in both mycelial types, it is probable that metabolites or lytic products in the pellets inhibit tentoxin-forming enzymes under submerged conditions.     

New vitreous matrix for chromium waste immobilization

Common waste glasses (window, bottle glass or tableware) with fly ash form a glass matrix for chromium waste immobilization. Soluble chromium from residual waters was adsorbed on fly ash; the resulting solid contained 23.7% Cr⁶⁺. The three glass wastes, chromium-containing fly ash, and borax were used to make glasses in weight ratios waste glass: borax: fly ash of 1: 1: 1 and 1.5: 0.5: 1. The hydrolytic stability ranged from 18.46 to 28.13 µg g^?1 soluble Na₂O, qualifying them in the HGB1 class. The chemical stability, characterized by the dissolution rate, was 0.011–0.077 µg cm^?2 h^?1, depending on the glass composition and the aggressive medium pH. The chromium leachability is influnced by the glass composition and the pH of the leaching solution, ranging between 0–0.015% of the total chromium. Chromium waste vitrification is a viabile solution with multiple economic advantages.

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Nanoparticles synthesis by electron beam radiolysis

Electron beam (EB) irradiation is a useful method to generate stable silver nanoparticles without the interference of inherent impurities generated from chemical reactions. Our experiments were carried out using linear electron beam accelerators with two different EB absorbed dose rates: 2 kGy min^?1 and 7–8 kGy s^?1, and with different absorbed dose levels. The optimum conditions for silver nanoparticles (AgNPs) generation by radiolysis, or by radiolysis combined with chemical reduction, were established. In order to obtain a good yield for AgNPs synthesized by radiolysis, a high dose rate is required, resulting in a rapid production process. At low absorbed dose rates, the utilization of a stabilization agent is advisable. By modifying the experimental conditions, the ratio between the chemical and radiolytic reduction process can be adjusted, thus it is possible to obtain nanoparticles with tailored characteristics, depending on the desired application.

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Microwave assisted extraction of essential oils from enzymatically pretreated lavender (<Emphasis Type="Italic">Lavandula angustifolia</Emphasis> Miller)

In this study, microwave assisted a hydrodistillation process (MWHD) of essential oils from lavender (Lavandula angustifolia Miller) was investigated. In order to examine any potential differences in essential oil extraction, the lavender flowers underwent enzymatic pretreatment.A 2³ factorial design of experiments, combined with statistical methods of data analysis were used to optimize enzymatic pretreatment and to evaluate the influence of major variables (enzyme concentration, temperature and pH) on the performance of the microwave assisted extraction.Under optimal conditions, an extraction yield of 24 mg oil g^?1 substrate was achieved (an increase by approximately 25% in comparison with the classic extraction conditions of conventional hydrodistillation).The main compounds of the essential oils obtained were analyzed and identified by gas chromatography coupled to mass spectrometry (GC-MS). Analyzing the data obtained indicated that the content of main compounds (linalool and linalyl acetate ? 73%) was greater than that obtained by conventional extraction (67%).

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Obtaining and characterizing modified tannins by physical-chemical methods

After oxidation of tannins (Tannins 1–3) extracted from grape seeds, Tanoxil compounds with increased solubility and enhanced antioxidant activities were synthesized. In this research, ABTS^+· assay and a chemiluminescence method were used for antioxidant measurement of hydrophilic compounds (Tanoxils 1–3).

It was observed that the antioxidant activity (AA, %) is very similar for the three samples, while at the same time AA is quite high (93.16%–96.48%). The percentage of inhibition by ABTS^+· is higher for Tanoxil 2 (96.4%) as compared to Tannin 2 compound (14.34%). Moreover, the total content (T_CF) of carboxyl and phenolic groups was investigated. T_CF values, determined for Tanoxil products, revealed a double (Tanoxil 1) or triple (Tanoxil 2) increase as compared to the value of the T_CF of Tannin 1. Tanoxil products represent an interest for future research as they have a high AA (96.4%) and the content of acidic groups is significant (T_CF, 0.191 meq g^?1).

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The use of ultrasonic treatment for improvement of metrological performance of sorption atomic absorption determination of lead(II) and cadmium(II) traces

The optimal parameters for ultrasonic treatment (frequency 200–300 kHz, intensity 2–4 W cm^?2) were obtained to intensify Pb(II) and Cd(II) sorption concentration by carbon sorbent from apricot pit. The combined action of ultrasonic frequency of 18 kHz and 1 MHz on concentrate slurry increases its sedimentation stability from 3 to 180 minutes and decreases S_r value up to 7% at Pb(II), Cd(II) hybrid sorption atomic absorption determination in natural waters, brines, common salt.      

Thermal oxidation of irradiated magnetic fluids and their component surfactants and dispersing oils

Magnetic fluid applications require stability under demanding conditions. Complete magnetic fluids and their component surfactants and dispersing oils were irradiated. Their subsequent thermal oxidation was characterized by chemiluminescence and DSC. Except for polyisobutylsuccinic anhydride, irradiation sensitized the components toward oxidation. The components were ranked by stability. Complete fluids were more stable than would be predicted from their components suggesting that they may be used for nuclear applications.

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