A rapid and sensitive ion chromatographic technique for the determination of sulfate and sulfate reduction rates in freshwater lake sediments

Abstract Newly developed low capacity columns were used in suppressed ion chromatography for rapid and highly reproducible determination of SO₄²⁻ in porewater samples from freshwater sediments without preconcentration of samples. With a 50 μl injection the detection limit for SO₄²⁻ was ca. 50 pmol (= 1 μ M) with a precision of 1–3% at the 10–200 μM level and <1% at concentrations above 200 μM. SO₄²⁻ could be measured in 4–5 min with the routinely used eluent (3.0 mM NaHCO₃/0.8 mM Na₂CO₃). When the strength of the eluent was increased to 3.0 mM NaHCO₃/2.0 mM Na₂CO₃, sulfate analysis was possible in less than 3 min, provided that samples were nitrate-free. Under these conditions S₂O₃²⁻ could also be sensitively determined in about 6 min. Examples of application of the method are given for measurements of sulfate reduction rates in freshwater sediment samples from Lake Constance.     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

SoxV,an orthologue of the CcdA disulfide transporter,is involved in thiosulfate oxidation in Rhodovulum sulfidophilum and reduces the periplasmic thioredoxin SoxW

Proteins of the CcdA/DsbD family have previously been found to be involved in the protein disulfide isomerase and cytochrome c maturation pathways of bacteria. SoxV is a CcdA homologue encoded by a genetic locus involved in lithotrophic thiosulfate oxidation in Rhodovulum sulfidophilum. Mutagenesis studies demonstrate an essential and specific role for SoxV in thiosulfate oxidation. Another protein encoded by the same locus, SoxW, is a periplasmic thioredoxin. SoxW was found to be in the reduced state during growth of R. sulfidophilum in the presence of thiosulfate. Maintenance of SoxW in the reduced state was shown to require SoxV. Nevertheless, SoxW was found to be dispensible for thiosulfate oxidation suggesting that SoxV reduces more than one periplasmic partner protein.     

Determination of cadmium,cobalt, copper,iron, manganese,and zinc in thyroid glands of patients with diagnosed nodular goitre using ion chromatography

The aim of this study was to estimate and compare the contents of selected metals in 65 pathological (diagnosed nodular goitre) and 50 healthy human thyroid tissues (taken during autopsies). Ion chromatography (IC) preceded by microwave mineralization was applied for the first time for determination of cadmium, cobalt, copper, iron, manganese, and zinc in human thyroid samples. The study proved that the concentrations of Cu²⁺, Mn²⁺, Fe³⁺, and Zn²⁺ were significantly higher in the control group (healthy thyroids) in comparison with the studied group (nodular goitre) (p < 0.05), whereas for Co²⁺ the difference between two means of concentration (healthy vs pathological thyroids) was not significant statistically at 0.05 significance level. Measurement accuracy was verified by measurements of NIST standard reference material (1566a Oyster Tissue). Very good precision (RSD below 5%) and recoveries (above 90%) were evaluated.     

Soil aggregates in a tropical deciduous forest: effects on C and N dynamics, and microbial communities as determined by t-RFLPs

The aim of this study was to analyze C and N dynamics, as well as, soil bacterial community structure within soil micro- and macro-aggregates in a tropical deciduous forest in México. We measured, for three landscape positions and three seasons of the year: total, microbial and available forms of C and N; potential C and N mineralization; and soil bacterial communities by using t-RFLPs. The highest total C concentrations were found in the north-slopes and in the dry season (DS) samples. In general, micro-aggregates had higher concentrations than macro-aggregates of available C and N forms, and microbial C. Similarly, micro-aggregates had the highest potential C mineralization and net N mineralization. We detected 149 different OTUs (operational taxonomic units) from which 50% was shared by the two aggregate size fractions, 25% was exclusive to micro-aggregates and the 25% left was found only in macro-aggregates. Top-hills were richer in OTUs than north and south-slopes. The Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis indicated clear differences in community composition between the two aggregate size-fractions in relation to the presence of OTUs. These results suggest that the main difference between micro- and macro-aggregates is due to the community structure within each soil fraction and this difference could affect soil nutrients dynamics.     

Control of Methane Metabolism in a Forested Northern Wetland,New York State,by Aeration,Substrates, and Peat Size Fractions

Although many northern peat-forming wetlands (peatlands) are a suitable habitat for anaerobic CH 4 -producing bacteria (methanogens), net CH 4 fluxes are typically low in forested systems. We examined whether soil factors (aeration, substrate availability, peat size fractions) constrained net CH 4 production in peat from a Sphagnum -moss dominated, forested peatland in central New York State. The mean rate of net CH 4 production measured at 24° C was 79 nmol g -1 d -1 , and the mean rate of CO 2 production (respiration) was 5.7 w mol g -1 d -1 , in surface (0 to 10 cm) and subsurface (30 to 40 cm) peat. Saturated peat (900% water content) exposed to oxic conditions for 2 days or 14 days showed no net CH 4 production when subsequently exposed to anoxic conditions. Rates of CO 2 production, measured concomitantly, were essentially the same under oxic and anoxic conditions, and net CH 4 consumption under oxic conditions was barely affected by short-term exposure to anoxic conditions. Therefore, methanogens were particularly sensitive to aeration. Net CH 4 production in whole peat increased within hours of adding either acetate, glucose, or ethanol, substrates that methanogens can convert directly or indirectly into CH 4 , indicating that availability of these substrate might limit net CH 4 production in situ. In longer incubations of 30 days, only ethanol addition stimulated a large increase in net CH 4 production, suggesting growth in the population of methanogens when ethanol was available. We fractionated peat into size fractions and the largest sized fraction (> 1.19 mm), composed mostly of roots, showed the greatest net CH 4 production, although net CH 4 production in smaller fractions showed the largest response to ethanol addition. The circumstantial evidence presented here, that ethanol coming from plant roots supports net CH 4 production in forested sites, merits more research.     

Bacteriological studies on the sulfur cycle in the anaerobic part of the hypolimnion and in the surface sediments of rotsee in Switzerland scientific report of the advanced course of microbial ecology, sponsored by FEMS and the Swiss society for Microbiology, held at Kastanienbaum, Switzerland, 12 September–9 October 1982

Abstract The microbial ecology of the sulfur cycle in the anaerobic part of Rotsee (Switzerland) was studied. Almost all the sulfate reduction took place at the sediment surface at a rate of 2 mmol SO²⁻₄ reduced m⁻² day⁻¹. Approx. 10⁴ sulfate reducers per ml were present in the surface sediments. The sulfide produced was phototrophically consumed mainly by Thiopedia rosea, Lamprocystis roseopersicina and ' Pelochromatium roseum ' consortia. Thiopedia rosea migrated diurnally about one meter. Bacterial photosynthesis was limited by light and sulfide rather than by temperature.     

Competition and coexistence of sulfate-reducing bacteria,acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol⁻¹, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol⁻¹, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.     

Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). HS-SPME was performed using a 75 microm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 degrees C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC-MS method was established over four orders of magnitude and the limit of detection was 0.005 microg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1-8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n=5; median, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.     

High-speed gel microelectrophoresis, a new and easy approach for detection of PCR-amplified microbial DNA from environmental and clinical samples in microgels using conventional equipment

AIMS: Microelectrophoresis allows the detection of DNA bands using minimal amounts of sample in a short time, but commonly requires the use of special equipment which is not available in all laboratories. This fact has limited the application of this technique in microbiology despite its advantages. In this work, we describe a new approach to perform gel microelectrophoresis, named high-speed gel microelectrophoresis (HSGME), and its application for rapid detection of bacteria, protozoa and viruses in clinical, vegetal and environmental samples. METHODS AND RESULTS: Aliquots of 0.4-1 microl of PCR product were loaded in 2 cm 1% agarose microgels and electrophoresed at high voltage (125 V cm(-1)) in conventional submarine horizontal mini-slabs. By using HSGME, single-DNA bands obtained after specific-PCR useful in diagnosis of different diseases caused by micro-organisms were detected in 5 min. CONCLUSIONS: HSGME is a rapid and easy procedure applicable to detection of microbial genes, which is carried out using conventional equipment and thus can be performed in any research and diagnostic laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The performance of HSGME saves up to 90% time, material and energy costs, as well as laboratory hazardous wastes including carcinogenic agents used for visualizing DNA bands.     

Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C₈ column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma. This method was used successfully for an investigation of the disposition of pemetrexed in patients receiving 500 mg/m² as a 10-min infusion.     

Determination of amino acids in foods by reversed-phase HPLC with new precolumn derivatives,butylthiocarbamyl, and benzylthiocarbamyl derivatives compared to the phenylthiocarbamyl derivative and ion exchange chromatography

New precolumn derivatizing reagents for analysis of amino acids by HPLC—butylisothiocyanate (BITC) and benzylisothiocyanate (BZITC)—reacted quantitatively with 22 standard amino acids and the amino acids in the acid hydrolysate of food and protein standard, bovine serum albumin (BSA), at 40°C for 30 min to yield butylthiocarbamyl (BTC) amino acids and at 50°C for 30 min to yield benzylthiocarbammyl (BZTC) amino acids. BTC and BZTC amino acids were successfully separated in 35 min on the reversed-phase Nova-Pak C18 column (30 cm × 3.9 mm, 4 μm). The optimum wavelengths for determination of BTC and BZTC derivatives were 240 nm and 246 nm, respectively. Analysis of the results obtained with BSA and food samples as BTC and BZTC derivatives showed good agreement with those determined as ion-exchange chromatography and data presented in the literature. The advantage of BITC reagent over the phenylisothiocyanate (PITC) and BZITC was that it had high volatility, so the excess reagent and by-products were easily removed in about 10 min, compared to about 1 h in the PITC and BZITC reagents. In the BTC and BZTC derivatives, cystine and cysteine were determined separately, but in the PTC amino acids derivatized with PITC reagent they were resolved into single peak.     

Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron. A simple and rapid HPLC–UV method for the separate determination of ICL670 and Fe–[ICL670]₂ in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4°C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C₁₈ column using 0.05 M Na₂HPO₄ and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25–20 μg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.     

The absolute concentration of nigral neuromelanin, assayed by a new sensitive method, increases throughout the life and is dramatically decreased in Parkinson’s disease

The concentration of neuromelanin (NM) in substantia nigra pars compacta (SNPC) has been measured in male and female normal subjects at different ages in the range 1–97 years old and in SNPC of parkinsonian patients. A very similar age trend of NM concentration was found in both sexes. In the first year of life NM was not detectable, between 10 and 20 years the NM levels were 0.3–0.8 μg/mg of SNPC, between 20 and 50 years were 0.8–2.3 μg/mg SNPC and between 50 and 90 were 2.3–3.7 μg/mg of SNPC. In parkinsonian subjects, the NM levels were 1.2–1.5 μg/mg of SNPC, which is less than 50% with respect to the age-matched controls. These data demonstrate a continuous NM accumulation in SNPC neurons during aging, the presence of large amounts of NM in SNPC and severe depletion of NM in Parkinson’s disease.     

Determination of a new podophyllotoxin derivative, TOP-53, and its metabolite in rat plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method was developed for the determination of a new podophyllotoxin derivative, TOP-53 (I), and TOP-53 glucoronide (II) as its major metabolite in rat plasma and urine. For the analysis of I, the sample was chromatographed on a reversed-phase C₁₈ column with electrochemical detection after consecutive two-step liquid-liquid extractions. Compound II was determined as I after enzymatic hydrolysis of II. This method was validated sufficiently with respect to specificity, accuracy, and precision. The limiits of quantitation for both I and II were 2 ng/ml in plasma and 10 ng/ml in urine. The method is thus useful for the pharmacokinetic study of I.     

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1–5 fmol/injection (S/N=3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C₁₈ column before conducting the labelling reaction. Pro–Hyp, Pro–Gly and Pro–Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5–4.8 and 1.7–5.8%, respectively. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human urine were 97.6±28.2, 2.74±1.48, 2.08±1.13, 6.71±3.34 and 2.30±1.59 nmol/mg creatinine, respectively.