A flow method with photocatalytic oxidation of dissolved organic matter using a solid-phase (TiO2) reactor followed by amperometric detection of consumed oxygen

A photocatalytic sensor for the determination of chemical oxygen demand (COD) using titanium dioxide, based on the use of a pair of oxygen electrodes and flow injection analysis, is described. The measuring principle is based on the direct determination of the oxygen concentration change resulting from photocataltic oxidation of organic compounds. One of the two oxygen electrodes, the reference oxygen electrode, was utilized to measure the reference signal responding only to oxygen present in the injected samples. Oxygen consumption due to the TiO2-catalyzed photochemical oxidation of organic compounds in samples was monitored with the working oxygen electrode. The COD value of this sensor was calculated as the difference of the currents at reference and working oxygen electrodes, respectively. The operation characteristics of the sensor are demonstrated using artificially treated wastewater as a substrate. The sensor was also applied to the determination of COD in real water samples from dam reservoirs (n = 20) all over Japan. The results were in good agreement with those from the conventional COD methods (i.e., permanganate and dichromate methods).     

Liquid mixing by large gas bubbles in bubble columns

Liquid mixing by large gas bubbles of spherical cup was investigated for co- and counter-current contact of air-water system with bubble columns of 5 and 10 cm dia. The results obtained are that for the column of 5 cm dia., the longitudinal dispersion coefficient ranges from 5 to 20 cm²/sec for superficial gas velocity from 0·07 to 8 cm/sec and that for the one of 10 cm in diameter it ranges from 9 to 45 cm²/sec for that from 0·035 to 8 cm/sec. Liquid mixing under the coexistence of large and small bubbles was also investigated and it was found that the gas holdup was fairly well explained by an equation derived on the assumption that the mixture of small bubbles and liquid behaves independently of large bubbles. The expansion model was applied to the experimental results on the longitudinal dispersion coefficient and it was observed that there should be the lower limit in the holdup of small bubbles where this model can be applied.     

Acidophilic xylanase from Aureobasidium pullulans: efficient expression and secretion in Pichia pastoris and mutational analysis

A yeast-like fungus Aureobasidium pullulans var. melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-1,4-beta-xylanase with an optimum pH of 2.0 [Ohta et al., J. Biosci. Bioeng., 92, 262-270 (2001)]. The xynI cDNA encoding the precursor protein (XynI) was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter. The 34 amino acid prepro-signal peptide of the A. pullulans XynI directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium. The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae alpha-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P. pastoris acid phosphatase. The N-terminal amino acid sequence and the apparent M(r) of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A. pullulans XynI signal sequence in P. pastoris. The three-dimensional model and mutational analysis of the xynI gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft, respectively, play a significant role in its low pH optimum.     

Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, alpha-1-fetoprotein and beta2-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.     

Surface properties of carbon micro-coils oxidized by a low concentration of oxygen gas

Vapor-grown carbon micro-coils were oxidized under a low O₂ flow-rate for introducing oxygen-containing functional groups on the surface. The surface characteristics were then examined. The O1s/C1s intensity ratios of the XPS spectra measured on the surface increased on using the oxidation treatment. The maximum O1s/C1s ratio of 11.4 at.% was obtained under the following conditions: (a) the flow-rate of the mixed gas of O₂+Ar (O₂/Ar=1/10) was 82.5 sccm; (b) the oxidation temperature was 600°C; and (c) the treatment time was 30 min. The maximum O1s/C1s ratio is about 3.5 times that of the source carbon coils. The specific surface area significantly increased due to the oxidation treatment and attained a maximum value of 1050 m² g⁻¹, which is about 10 times that of the source carbon coils. As the specific surface area increased, the surface morphology of the carbon coils became more complicated on a nanometer scale.     

Surfactant effects on hydrate formation in an unstirred gas/liquid system: An experimental study using HFC-32 and sodium dodecyl sulfate

This paper deals with the effects of a surfactant additive on the formation of a clathrate hydrate in a quiescent guest-gas/liquid-water system. The paper first presents our strong suspicion against the existing hypothesis that the surfactant-micelle formation in the liquid-water phase promotes the hydrate formation. It is pointed out that the Krafft point for sodium dodecyl sulfate (SDS), a popular anionic surfactant often used in previous hydrate-forming experiments, is presumably higher than the system temperatures set in these experiments and hence that no micelles may have formed in these experiments. The paper then describes our experimental observations of the hydrate formation from a hydrofluorocarbon gas, HFC-32 (CH₂F₂), to show how the hydrate formation behavior is affected by the addition of SDS to the water when brought into contact with HFC-32. In each experiment, HFC-32 gas was continuously supplied to a rectangular chamber partially filled with a quiescent pool of water (pure water or an aqueous SDS solution) to compensate for the gas consumption due to the hydrate formation, thereby maintaining a constant pressure inside the chamber. The present experiments featured the following characteristics: (a) detailed visual observations along horizontal axes through large side windows in the test chamber, and (b) surface tension measurements of the aqueous SDS solutions with the aid of a pendant-drop device inserted in the same chamber to determine the SDS-in-water solubility, which seems to have been misunderstood as the critical micelle concentration (CMC) in some previous studies, under the hydrate-forming conditions. The former revealed that the addition of SDS to the pool-forming water results in the formation of thick, highly porous hydrate layers not only on the liquid-pool surface but also on the chamber walls above the level of the pool surface, leaving the bulk of the liquid pool free from hydrate crystals. The latter led to an important finding that the SDS concentration at which the rate of the hydrate formation peaks is slightly lower than the solubility (the false CMC). An excessive addition of SDS beyond the solubility was found to cause a decrease in the rate of hydrate formation but an increase in the final level of the water-to-hydrate conversion.     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.