Reaction Flasks Research Articles

Petroleum-hydrocarbon utilization by native bacterial population from a wastewater canal Southwest Nigeria

The application of a consortium of native bacterial species in bioremediation processes has long been desired in Nigeria because they would be cost effective and efficient in terms of acclimation time. Two Nigerian crude oils (Bonny light and Escravos blend) were exposed to the wastewater canal via oil impregnated membrane filters (0.45 μm diameter) for 21 days in a microcosm experiment. Bacterial petroleum-hydrocarbon utilizers were later harvested from both the millipore membrane filters and laboratory biodegradative studies. Some of the striking discoveries made were that pH fluctuations in the reaction flasks were due to microbial activities, microbial enzymes, presence of crude oil degradation metabolites such as organic acids, surfactants and aldehydes. More petroleumhydrocarbon utilizers were detected at 0 to 15 cm depth than at 15 to30 cm. The petroleum sample with higher fractions of saturated hydrocarbon was biodegraded faster (Bonny light) than the ones with higher fractions of asphaltenes and aromatics (Escravos blend). Escravos blend had C-14 component undegraded after the 3-week oil explosure to the waste water. The physico-chemical properties of the freshwater ecosystem were determined and it supported previous conclusion on the latent self purification capability of the aquatic ecosystem inspite of frequent oil pollution incidents. The mean temperatures of the freshwater ecosystem were within the mesophilic range 27-29°C and the pH of the environment supported acidophilic bacterial consortium. Gas chromatographic profiles of the mineralization process of Bonny light and Escravos blend gave the conclusive evidence for the capability of the native bacterial population to mineralize petroleum hydrocarbons in wastewater, at optimum physico-chemical conditions in the habitat. Petroleum-hydrocarbon utilizers detected included Bacillus megaterium , Pseudomonas putida , Micrococcus luteus , Bacillus brevis , B. punilis and Enterobacter aerogenes . Keywords: Biodegradation, bioremediation, petroleum-hydrocarbon

Acquisition of Fe from Various Natural Organic Matter Isolates by Aerobic Pseudomonad Bacteria

Iron (Fe) is an essential nutrient to most microorganisms. Aerobic microorganisms exhibit various strategies for acquiring Fe at near-neutral pH conditions, where Fe oxyhydroxides are insoluble. Although much research has focused on microbial acquisition of Fe from minerals, little is known about Fe acquisition from natural organic matter (NOM). Yet, in surface waters, soils and shallow sediments, Fe is often associated with natural organic matter (NOM), and this NOM-associated Fe could represent an important pool of Fe for microorganisms. Here, we investigated the growth of aerobic Pseudomonas mendocina on soil and surface water NOM samples containing Fe, under Fe-limited conditions. In the presence of NOM, bacteria grew to population sizes greater than in no-Fe-added controls, indicating that the bacteria were able to access Fe associated with NOM. Maximum population size correlated with the NOM-associated Fe concentration. In an additional experiment, Pseudomonas putida was able to acquire Fe from an NOM sample, demonstrating that this ability is not limited to P. mendocina. When Fe was added as 30 μ M FeEDTA plus NOM, together in the same reaction flasks, P. mendocina and P. putida growth was less than in the presence of 30 μM FeEDTA alone. The fact that Fe sources are not simply additive and that the presence of NOM inhibits growth in FeEDTA suggests that further study on the responses of bacteria to a combination of Fe sources is needed to understand the complexities of bacterial Fe acquisition in the subsurface.

Polymer Supported Calix[4]arene Schiff Bases: A Novel Chelating Resin for Hg2+ and Dichromate Anions

This article describes the synthesis and characterization of two new calix[4]arene Schiff bases and their polymeric resins. The extraction properties of these “proton switchable extractants” with alkali, transition, post transition metal cations and for dichromate anions are reported. The two new calix[4]arene based Schiff bases (5 and 6) have been synthesized from 5,17‐diformyl‐25,27‐dipropoxy‐26,28‐dihydroxycalix[4]arene (4) by treatment with 3‐amino‐methylpyridine and 1,8‐diaminooctane in two separate reaction flasks following the same procedure. Compounds 5 and 6 have been appended to a polymeric resin by treatment with Merrifield resin through a nucleophilic substitution reaction. The receptor compounds (3 and 5–8) do not extract alkali metal cations, but show some selectivity toward transition metal cations, and a particularly high selectivity to Hg2+ and Pb2+. The protonated forms of all of the calixarene‐based receptors are good extractants for transferring Cr2O7 2−/HCr2O7 − anions from an aqueous into a dichloromethane layer.

Small Cyclic Disulfide Peptides: Synthesis in Preparative Amounts and Characterization by Means of NMR and FT‐IR Spectroscopy.

Two cyclic disulfide-bridged tetrapeptides [cyclo(Boc−Cys−Pro−Aib−Cys−OMe) and cyclo(Boc−Cys−Pro−Phe−Cys−OMe)] were prepared in high yield and purity. The use of double-walled reaction flasks, which were attached to an external cryostat, gave perfect temperature control of the reaction. The acetamidomethyl and trityl group were used for the protection of the thiol groups of Cys. Disulfide bond formation was obtained by cleavage of the protection groups and subsequent oxidation of the free thiols with iodine under high dilution conditions in one step. The obtained cyclic peptides were checked for purity by analytical HPLC, and identified by NMR spectroscopy and a variety of standard analytical methods (IR, m. p., FAB-MS, ELA, TLC). Conformational properties of the peptides were derived from one- and two-dimensional NMR experiments. Temperature-dependent NMR and FT-IR experiments allowed for the determination of the degree of inter- and intramolecular hydrogen bonding of the cyclic tetrapeptides. The results from the temperature-dependent NMR experiments are in good agreement with the observed dynamics of the peptides in the amide I and II region, which were determined by means of temperature-dependent FT-IR spectroscopy using the ATR technique. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Small Cyclic Disulfide Peptides: Synthesis in Preparative Amounts and Characterization by Means of NMR and FT‐IR Spectroscopy

AbstractTwo cyclic disulfide‐bridged tetrapeptides [cyclo(Boc−Cys−Pro−Aib−Cys−OMe) and cyclo(Boc−Cys−Pro−Phe−Cys−OMe)] were prepared in high yield and purity. The use of double‐walled reaction flasks, which were attached to an external cryostat, gave perfect temperature control of the reaction. The acetamidomethyl and trityl group were used for the protection of the thiol groups of Cys. Disulfide bond formation was obtained by cleavage of the protection groups and subsequent oxidation of the free thiols with iodine under high dilution conditions in one step. The obtained cyclic peptides were checked for purity by analytical HPLC, and identified by NMR spectroscopy and a variety of standard analytical methods (IR, m. p., FAB‐MS, ELA, TLC). Conformational properties of the peptides were derived from one‐ and two‐dimensional NMR experiments. Temperature‐dependent NMR and FT‐IR experiments allowed for the determination of the degree of inter‐ and intramolecular hydrogen bonding of the cyclic tetrapeptides. The results from the temperature‐dependent NMR experiments are in good agreement with the observed dynamics of the peptides in the amide I and II region, which were determined by means of temperature‐dependent FT‐IR spectroscopy using the ATR technique. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

The synthesis of metoprolol monitored using Raman spectroscopy and chemometrics

The synthesis of Metoprolol base was studied using Raman spectroscopy with a 785-nm laser, optical fibres, a holographic transmission grating, confocal optics and a charge-coupled device (CCD) detector. The reaction mixture was heated according to a temperature gradient and spectra of the reaction mixture were obtained by focusing the laser beam through ordinary reaction flasks. Because of overlapping bands, multivariate techniques such as principal components analysis (PCA) and partial least-squares projections to latent structures (PLS) were used in the evaluation of the obtained spectra. The use of PCA or PLS against time does not require any calibration samples and a quantitative calibration is not necessary in order to monitor the reaction. A method for reaction endpoint determination, based on euclidean distances in the score space, is presented. The use of multivariate batch control charts have been demonstrated and a number of problems and solutions regarding the sample presentation have been discussed. The effect of spectral pretreatment on the multivariate results is shown and discussed. The monitoring results show that the time to produce Metoprolol base could be reduced.

A Versatile Fume Hood Support To Secure Large Reaction Flasks

We describe a simple support that can be used with 12 or 22 L reaction flasks. Common fume hoods readily accommodate reaction vessels up to 5 L by attachment to a lattice in the rear. Anchoring larger glassware using this relatively frail lattice arrangement results in problems with bottom drop valves, access to apical openings, and support for large masses. The apparatus described herein permits the safe and inherently stable support of jacketed 12 or 22 L reactor vessels.

Soil Depth and Temperature Effects on Microbial Degradation of 2,4‐D

AbstractNumerous soil factors and climatic conditions affect the degradation rate of pesticides in soils. A major soil factor influencing herbicide degradation is the composition and abundance of the microbiota, which has been shown to vary considerably with soil depth. Another important variable affecting microbial growth and degradation kinetics is temperature. Soil samples from 0‐ to 30‐, 30‐ to 60‐, and 60‐ to 120‐cm depths of two Montana soils were placed in reaction flasks and treated with 14C‐labeled 2,4‐D at representative field use rates at temperatures of 10, 17, and 24°C. A carrier gas was used to continuously evacuate evolved 14CO2 into NaOH traps as a measure of 2,4‐D degradation. Comparisons of the effects of soil depth and temperature were made by fitting experimental data to both first‐order and logistic kinetic models. Degradation rates of 2,4‐D decreased significantly with increasing soil depth and were positively correlated with bacterial plate counts. Effects of temperature on degradation rate constants were adequately described using the Arrhenius equation. Degradation rates of 2,4‐D and bacterial enumerations were positively correlated with changes in soil organic C as a function of soil depth. These results support the idea that changes in organic C with soil depth could be used as a parameter for estimating changes in degradation rate as a function of soil depth. Efforts to model the transport of 2,4‐D in soils should account for variation in degradation rate as a function of soil depth and temperature.

Biodegradation of Trichloroethylene in Finished Compost Materials

Growing interest in the use of biofiltration technology to remove toxic organic compounds from gaseous waste streams has led to the investigation of various solid packing materials to support microbial populations capable of contaminant biodegradation. Finished compost material has been used as a biofiltration packing matrix for the treatment of noxious odors and the removal of several gaseous organic contaminants. Trichloroethylene (TCE), a widespread groundwater contaminant, has been shown to undergo aerobic biodegradation under a variety of environmental conditions. The current investigation focused on the capacities of five different finished compost materials to remove TCE from head space vapors in small reaction flasks. Due to the cometabolic nature of aerobic TCE biodegradation, enrichment of compost materials with propane or methane as primary substrates was tested as a means to stimulate biological TCE removal. Results indicate that all of the materials tested removed at least 85 percent of the a...

Automated synthesis of radiopharmaceuticals for positron emissiontomography: an apparatus for labelling with [11C] methyl iodide (MIASA)

A fully automated apparatus for the routine synthesis and formulation of short-lived 11C (t1/2 = 20 min) labelled radiopharmaceuticals for positron emission tomography (PET) has been developed. [11C]Carbon dioxide is converted to [11C]methyl iodide, which can be used to label a wide variety of substrates by methylation at C, N, O, or S electron rich centres. The apparatus, MIASA (methyl iodide automated synthesis apparatus), was designed to operate as part of an automated labelling system in a shielded ‘hot’ laboratory. The apparatus was designed without the size constraints of typical instrumentation used in hot cells, although it is compact where necessary. Ample use of indicators and sensors, together with compact design of the reaction flasks for small dead space and efficient evaporation, led to good reliability and performance. The design of the hardware and software is described in this paper, together with a preparation of 3-N-[11C]methylspiperone as a sterile injectable solution in physiological saline.

Stimulation by lepidocrocite (7-FeOOH) of Fe(II)-dependent nitrite reduction

Fe(II)-dependent nitrite reduction was studied in oxygen-free reaction flasks at pH values from 6.0 to 8.5 and in the presence and absence of the Fe(III) oxyhydroxide lepidocrocite (γ-FeOOH). When ionic Fe 2+ was added to flasks containing lepidocrocite, rapid Fe 2+ consumption and release of H + took place. The solid phase turned dark and contained magnetite (Fe 3O 4). This reaction was independent of the presence of NO 2 −. However, rapid NO 2 − reduction primarily to N 2O was observed only after the Fe 2+ binding to lepidocrocite was initiated; insignificant NO 2 − reduction was observed in control flasks without lepidocrocite. Both Fe 2+ binding and associated H + release and NO 2 − reduction were negligible at pH values of 7.0 and below, but increased dramatically as pH was raised to 8.5. The Fe(II) associated with a reactive complex formed during Fe 2+ binding to lepidocrocite, and not the ionic Fe 2+, seemed responsible for the reduction of NO 2 −. The catalytic effect of Fe(III) oxyhydroxide may stimulate Fe(II)dependent formation of N 2O from NO 2 − (chemodenitrification) in geochemical systems such as sediments and subsoils.

O6-methylguanine-DNA methyltransferase activity in tissues and cells of the rat respiratory tract

A product of alkylating agents and DNA, O6-methylguanine (O6-MG), can mispair with thymine, resulting in initiation of a carcinogenic tissue response. O6-alkylguanine-DNA alkyltransferase (AGT) is an acceptor protein responsible for repairing O6-MG. The purpose of our experiments was to characterize in vitro AGT activity in tissues and cells in the respiratory tract, a target tissue for inhaled alkylating agents. Anatomically defined regions throughout the respiratory tract of male F344 rats were obtained. These included two regions of the lateral wall of the left and right nasal airway (maxilloturbinates and ethmoturbinates), trachea, extrapulmonary bronchi and peripheral lung. Alveolar type II cells were also used in these studies. Radioactive 3H-methylated DNA was synthesized for use in all experiments. Removal of [3H]methyl from O6-MG was measured by high-pressure liquid chromatography after incubation for up to 30 min of tissue and cell extracts with the [3H]DNA. With the exception of tracheal and bronchial extracts, all tissues and cells analyzed contained AGT activity, which was found to increase proportionally to the amount of protein added to reaction flasks. AGT activity in tracheal and bronchial extracts was only detected at the highest protein concentration used (1.5 mg protein/ml) and ranged from 10–15 fmol/mg protein. AGT activity was highest in the lung (∼75 fmol/mg protein) and a region of the nasal tissue, the ethmoturbinates (∼45 fmol/mg protein). AGT activity in the maxilloturbinates was about 50% less than the AGT activity measured in the ethmoturbinates. These data suggest that methylated DNA in specific regions of the rat respiratory tract should be readily repaired, albeit to different extents.

Metabolism of 1,3-butadiene by lung and liver microsomes of rats and mice repeatedly exposed by inhalation to 1,3-butadiene

1,3-Butadiene, a colorless gas widely used as an intermediate in the production of synthetic rubber, is carcinogenic in rats and mice. Species differences exist in the sensitivity to inhaled 1,3-butadiene and the target tissue specificity for tumor formation. We examined whether repeated inhalation exposure of rats and mice to 1,3-butadiene would affect the rate of metabolism of 1,3-butadiene by lung and liver microsomes in these species. Male Sprague-Dawley rats and B6C3F 1 mice were exposed nose-only to air (control) or 7600 ± 170 ppm 1,3-butadiene (13 600 ± 300 μg/1) and 740 ± 10 ppm 1,3-butadiene (1300 ± 20 μg/1), respectively, for 6 h/day for 5 days. After the last exposure, nasal tissue (rats only), lungs and livers were removed from the animals and microsomes were prepared. Microsomes from the different tissues were incubated with 6 μmol 1,3-butadiene and 10 μmol NADPH for 30 min and the rate of disappearance of 1,3-butadiene from the reaction flasks was quantitated. There was a statistically significant ( P < 0.05) depression in the rate of 1,3-butadiene metabolism (50%) in microsomes from lungs of both rats and mice that were exposed repeatedly to 1,3-butadiene compared to control animals. There was no effect of repeated 1,3-butadiene exposure on liver or nasal tissue (rats only) metabolism of 1,3-butadiene in rats or mice. The data from these studies indicate that it is unlikely that species differences in sensitivity or tissue susceptibility are due to an inductive or inhibitory effect of 1,3-butadiene on its own metabolism in the tissues examined.

Apparent and real reducing ability of polypropylene in cold-vapour atomic-absorption spectrophotometric determinations of mercury

Experiments undertaken with polypropylene reaction flasks in connection with the Perkin-Elmer MHS-1 Mercury/Hydride System show that polypropylene very quickly adsorbs tin(II) chloride, which is not then removed by usual routine rinsing. This can cause mercury to be lost rapidly from solutions stored in uncleaned flasks as well as giving signals for mercury in coldvapour atomic-absorption methods even if reductant is not added. This apparent ability of polypropylene to reduce mercury(II) is rapid and ceases with careful cleaning of the apparatus. This apart, the polypropylene reaction flasks have a real, relatively slow, but considerable ability to reduce mercury(II), which was especially conspicuous if solutions without preservatives were agitated in them.

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in chicken aortas: Monooxygenation, bioactivation to mutagens, and covalent binding to DNA in vitro

The capacity of enzymes present in chicken aortas to catalyze the conversion of polynuclear aromatic hydrocarbons to metabolites that covalently bind to DNA and produce mutations in Salmonella typhimurium auxotrophs is described. Chicken aortic homogenates contained active monooxygenases capable of catalyzing the conversion of benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) to oxidized products as measured by a highly sensitive radiometric assay and high-pressure liquid chromatography. BaP was converted primarily to phenols, while DMBA was converted to hydroxymethyl, phenolic, and diol metabolites. Pretreatment of chickens with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (5,6-BF) resulted in significant ( p < 0.05) increases in the aortic oxidative metabolism of both BaP and DMBA; phenobarbital (Pb) and pregnenolone 16α-carbonitrile (PCN) were ineffective as inducers of chicken aortic monooxygenation. Chicken aortic S-9 homogenates catalyzed the bioactivation of BaP and DMBA to mutagens for S. typhimurium strains TA-98 and TA-1538. Aroclor 1254, 3-MC, 5,6-BF, Pb, or PCN pretreatments did not significantly enhance detectable mutagenesis. When aortic homogenates from untreated chickens were utilized, low specific activities of bound products of BaP, DMBA, or 3-MC to DNA were observed. Pretreatment of chickens with either Aroclor 1254 or 3-MC resulted in 7- to 12-fold increases in specific binding activity. Inhibition of DMBA and BaP metabolism as well as DNA binding was observed when 7,8-benzoflavone (130 μ m) or β-estradiol (100 μ m) were added to incubation flasks. 1,2-Epoxy-3,3,3-trichloropropane (13 μ m) enhanced binding of [ 3H]BaP and [ 3H]DMBA to DNA. Additions of glutathione to reaction flasks inhibited 3H binding to DNA, suggesting an important role of aortic nonoxidative metabolism of polycyclic aromatic hydrocarbons.

Flame emission studies of several metal chlorides with vapor-phase sampling

A method is described for the production of spectra of aluminum chloride, iron(III) chloride, silicon tetrachloride and titanium tetrachloride vapors by flame emission spectrometry. The aluminum and iron(III) chloride vapors were prepared by heating solid samples in reaction flasks; silicon and titanium tetrachlorides have sufficient vapor pressures at ambient temperatures to produce vapor-phase samples. Techniques have been developed to introduce the sample into the flame as a vapor, and to accommodate a large concentration of sample while requiring minimum preparation. Spectra were obtained individually and as a mixture over 240–600 nm. The analytical wavelength was chosen for each element, and 10-s integrations were made by utilizing a microcomputer to slew rapidly to the line of interest, hold for 10 s on the emission line, move off wavelength, hold for a 10-s background measurement and slew rapidly to the next line of interest. The microcomputer was also used to digitize and display the number of photons counted.

Stimulation by hematin of monooxygenase activity in extra-hepatic tissues from rats, rabbits and chickens

Micromolar quantities of hematin, when added to reaction flasks containing homogenates of extra-hepatic tissues, benzo(a)pyrene, and cofactors necessary for monooxygenation, caused up to a 40-fold enhancement of aryl hydrocarbon (benzo(a)pyrene) hydroxylase (EC 1.14.14.2) activity. Pretreatment of rats with 3-methylcholanthrene caused a reduction in the stimulatory effect of hematin. Metabolic profiles of 7,12-dimethylbenz(a)anthracene, obtained with high pressure liquid chromatography, illustrated the enhancement of monooxygenase activity by hematin at methyl carbons. It is speculated that the mechanism of the hematin effect is the reconstitution of a small pool, or pools, of apo-(cytochrome P-450) which exist in these extra-hepatic tissues, resulting in the formation of functional holoenzyme.

Differential inhibition of androst-4-en-3,17-dione aromatization by carbon monoxide in the presence of estr-4-en-3,17-dione

The aromatization of androst-4-en-3,17-dione or 17β-hydroxyandrost-4-en-3-one (testosterone) is not inhibited by carbon monoxide under normal incubation conditions, whereas the aromatization of corresponding 19-nor steroids (estr-4-en-3,17-dione and 17β-hydroxyestr-4-en-3-one) is readily inhibited under the same conditions. A possible explanation was found when it was shown that androst-4-en-3,17-dione and testosterone could displace bound carbon monoxide from human placental microsomal cytochrome P-450. The 19-nor steroids did not displace carbon monoxide, even at very high concentrations. These C-18 compounds appeared to facilitate complex formation and reversed the effects of the C-19 steroids. A mutual antagonism was observed with regard to effects on the formation of the carbon monoxide-cytochrome complex and opposing actions could be titrated. These observations suggested that the aromatization of androst-4-en-3,17-dione should be inhibited by carbon monoxide if sufficient concentrations of the 19-nor steroids were present in reaction flasks. This hypotheses was tested and positive results were obtained, providing strong evidence for the involvement of cytochrome P-450 in normal estrogen biosynthesis.

A Respirometer Technique for Evaluating the Potential Effects of Rapid Thermal Changes to Small Aquatic Organisms

A 20-channel Gilson differential respirometer has been modified to evaluate potential physiological adjustments of small aquatic animals exposed to rapid thermal changes. Time-temperature conditions can be achieved which are typical of large volume steam electric generating stations with once-through cooling systems terminating in submerged discharges. In entrainment studies the modified respirometer has been used for determining pre-stress oxygen consumption, simulation of a time-temperature curve, followed by subsequent post-stress oxygen consumption data. All reaction flasks remain connected to the volumometers throughout the entire operation, thereby avoiding the necessity of removing and resealing the reaction flasks to the connectors. This also eliminates the problem of transferring organisms in and out of the reaction flasks during a study.

Inactivation of erythropoietin by hepatic lysosomes.

SummaryStudies were performed to determine the effects of liver homogenates and subcellular fractions on the inactivation of ESF. ESF was assayed using the percentage of 59Fe incorporation into the erythrocytes of exhypoxic polycythemic mice. Liver inactivation of ESF was found to correlate with the activity of β-glucuronidase, a lysosomal marker enzyme. Incubation of ESF with hepatic lysosomal fractions resulted in a reduction in erythropoietic activity which was significant after 15 min. Further progressive inactivation occurred over a period of 6 hr. Decreasing the content of the lysosomal protein in the reaction flasks or heat denaturation of the lysosomal fraction substantially reduced the amount of ESF inactivation, providing additional evidence that this process is enzymatic.The authors acknowledge the excellent technical assistance of Mr. Jesse Brookins, Mr. Rene Stiaes and Miss Pat Dargon.