Characterization of a methane-utilizing strain and its application for monitoring methane.

AIMS: To explore new resources of methane-utilizing micro-organism and develop a microbial biosensing system for monitoring methane released from natural and semi-natural ecosystems. METHODS AND RESULTS: A methane (CH(4))-utilizing bacterial strain was isolated from paddy soil using CH(4) as the sole carbon source and identified as Klebsiella sp. ME17 by phenotyping and 16S rDNA sequence analysis. The efficiency of CH(4) utilization of strain ME17 was 83.2% by gas chromatography analysis. A microbial biosensing system for CH(4) detection was developed by combining immobilized cells of strain ME17 with a dissolved oxygen sensor. It was found that response time of the system to CH(4) was <90s. The dissolved O(2) consumption increased with increasing CH(4) from 0% to 16.0% (v/v) demonstrating a positive linear relationship with a low detection limit of 0.2% (v/v). The relative standard deviation is 3.48%. CONCLUSIONS: Klebsiella sp. ME17 isolate is capable of utilizing CH(4). The microbial biosensing system of strain ME17 has been successfully applied to measure standard CH(4) sample with satisfactory results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that certain strains of Klebsiella genus are capable of utilizing CH(4). Our proposed method appears very attractive for CH(4) measurement in coal mine.

Retention behaviour and fluorimetric detection of procaine hydrochloride using carboxymethyl-beta-cyclodextrin as an additive in reversed-phase liquid chromatography.

The retention behaviour of procaine hydrochloride on an Alltima octadecyl silica (C18) column, with a mobile phase containing negatively charged carboxymethyl-beta-cyclodextrin (CM-beta-CD), influenced by a combination of hydrophobic and electrostatic interactions was systematically investigated. Various factors affecting the retention of procaine on the C18 column such as the concentration of CM-beta-CD, pH and the methanol percentage in the mobile phase, were studied. An equation was applied to estimate the apparent binding constant of the CM-beta-CD-procaine inclusion complex as an aid for understanding the retention mechanism. The first analytical application of CM-beta-CD as a mobile phase additive for the determination of procaine was developed. The calibration curve was linear in the range 22-1360 ng ml(-1) with an RSD of 2.1%. The detection limit based on 3sigma was 1 ng ml(-1) with fluorimetric detection at the excitation and emission wavelengths of 305 nm and 350 nm, respectively. The limit of quantitation based on 10sigma was 22 ng ml(-1). The proposed method has been successfully applied to real sample analysis.

Determination of airborne formaldehyde by active sampling on 3-methyl-2-benzothiazolinone hydrazone hydrochloride-coated glass fibre filters.

Formaldehyde was sampled with the use of a standard miniature glass fibre filter coated with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). The formaldehyde hydrazone formed [i.e., the adduct of formaldehyde (HCHO) and MBTH] was desorbed from the filter with water and then oxidised by an iron(III) chloride-sulfamic acid solution to form a blue cationic dye in acidic medium which was subsequently determined by visible absorption at 628 nm. The recovery of HCHO as the cationic dye from MBTH-coated filters is 87-102% in the range 0.065-2.9 micrograms of HCHO. This corresponds to 4.3-193.3 micrograms m-3 in a 15 L air sample. The collection efficiency of the MBTH-coated filter is higher than 90%. When the filter sampling system is used in active mode, air can be sampled at a rate of up to 1 L min-1, affording an overall sensitivity of about 3 micrograms m-3, corresponding to about 2 ppb v/v HCHO at 1 atm and 273 K. The method was successfully applied to the determination of HCHO in samples of indoor and outdoor air with satisfactory results.

Restriction fragment length dimorphism-PCR method for the detection of extended-spectrum beta-lactamases unrelated to TEM- and SHV-types.

The diagnostic ability of the restriction fragment length dimorphism-polymerase chain reaction (RFLD-PCR) method was evaluated. Seven primer pairs, newly designed from 44 beta-lactamase genes encoding extended-spectrum beta-lactamases not related to TEM- and SHV-types, were used to differentiate OXA-2, FOX-3, CMY-3, IMP-1, and IMI-1 beta-lactamases. The RFLD-PCR was carried out successfully, and these genes were differentiated by the sizes of their PCR products and by the difference in restriction fragment length when each amplicon was digested with a unique restriction enzyme. This discriminatory detection of the genes was confirmed by sequencing the PCR products.

Determination of cyclamate in low-calorie foods by high-performance liquid chromatography with indirect visible photometry.

A rapid and simple method using reversed-phase high-performance liquid chromatography combined with indirect visible photometry at 433 nm was developed to determine cyclamate in some food samples. Cyclamate was not detected in these chosen samples as its use is banned in Hong Kong. Cyclamate can easily be detected in spiked samples using a mobile phase consisting of 30 mumol dm-3 Methyl Red and 0.02 mol dm-3 phosphate buffer (pH 7.0)-methanol in a volume ratio of 3:2. The column temperature was set at 23 degrees C. The detection limit was 0.14 mmol dm-3 and the relative standard deviation of the peak area response was 0.58% for a solution containing 5.0 mmol dm-3 of cyclamate (n = 8). This method was successfully applied to the analysis of eight spiked food samples and the cyclamate recoveries for these samples ranged from 93 to 99%.

Optode Membrane for Determination of Nicotine via Generation of Its Bromoethane Derivative.

A plasticized poly(vinyl chloride) optode membrane incorporated with a valinomycin ionophore, a H(+)-selective chromoionophore (ETH 5294), and a lipophilic potassium tetrakis(4-chlorophenyl)borate was used as a reversible sensing device for the indirect optical determination of nicotine. Nicotine was extracted from a tobacco product (1-5 g) and converted to its bromoethane derivative (NBD(+)Br(-)) by reacting with a solution of bromoethane in ethanol. NBD(+)Br(-) in a solution of 0.05 M boric acid-Borax buffer and 0.2 mM Triton X-100 was extracted into the bulk of the membrane and subsequently caused changes in optical absorption of the sensing layer. The response slope, dynamic working range, detection limit, sensitivity, selectivity, effects of buffer solution and neutral surfactant Triton X-100, and lifetime were discussed in detail. The response was pH dependent. At pH 8.5, the detection range was extended from 0.4 µM to 1 mM. Typical response times (t(95)) of the samples were 2-4 min. The optode method was successfully used to detect nicotine in a tobacco sample from the market (average content 0.720%; RSD 0.044%; n = 11). The interference of K(+) on the optode method can be prevented by the pre-extraction procedure. Malic acid and citrate showed no interferences. The recovery of nicotine as NBD(+) was 84-119% in the range 0.035-5% nicotine. The result was satisfactory compared with an AOAC UV standard method.

Alcohol sensing membrane based on immobilized ruthenium(II) complex in carboxylated PVC and surface covalently bonded alcohol oxidase.

Alcohol sensing membranes coated on overhead transparency films for the continuous monitoring of ethanol, propanol and butanol are presented. Alcohol oxidation catalyzed by alcohol oxidase in conjunction with the fluorescence quenching reaction of oxygen-sensitive dye ion-pair, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) didodecylsulphate was chosen for the determination. Alcohol oxidase was immobilized covalently on a plasticized carboxylated poly(vinyl chloride) membrane and the oxygen-sensitive dye ion-pair was entrapped in the same membrane. The sensing membrane relates oxygen consumption, as a result of enzymatic oxidation, to alcohol concentration. Measurements have been performed in air-saturated alcohol standard solutions of pH 7.0. Storage stability, reproducibility and the effect of pH on sensing membrane performance have been studied in detail. The alcohol sensing membrane proposed here is simple to prepare and has a fairly rapid response time of <1 min. It has been successfully applied to the determination of the ethanol contents in various spirits.