Evaluation of isotopic enrichment factors for the biodegradation of chlorinated ethenes using a parameter estimation model: toward an improved quantification of biodegradation

A model was developed to predict the concentrations of chlorinated ethenes and ethene during sequential reductive dechlorination of tetrachloroethene (PCE) from stable carbon isotope values using Rayleigh model principles and specified isotopic enrichment factors for each step of dechlorination. The model was tested using three separate datasets of concentration and isotope values measured during three experiments involving the degradation of PCE to vinyl chloride (VC), trichloroethene (TCE) to ethene, and cis-1,2-dichloroethene (cDCE) to ethene. The model was then coupled to a parameter estimation method to estimate values for the isotopic enrichment factors of TCE, cDCE, and VC when they are intermediates in the dechlorination to ethene. The enrichment factors estimated for TCE and cDCE when they were intermediates in biodegradation experiments were close to or within the published range of enrichment factors determined from experiments where TCE or cDCE were the initial substrates. In contrast, the enrichment factors determined by parameter estimation for experiments in which VC was an intermediate in biodegradation experiments were consistently more negative (by approximately 10 per thousandth) than the most negative published enrichment factor determined from experiments where VC was the initial substrate. This finding suggests that the range of enrichment factors for VC dechlorination may not be as narrow as previously suggested (-21.5 per thousandth to -26.6 per thousandth) and that fractionation during VC dechlorination when VC is an intermediate compound may be significantly larger than when VC is the initial substrate. These findings have important implications both for the current practice of extrapolating laboratory-derived isotopic enrichment factors to quantify biodegradation of chlorinated ethenes in the field and for understanding the details of enzymatic reductive dechlorination.     

Novel method for quantification of individual free fatty acids in milk using an in-solution derivatisation approach and gas chromatography-mass spectrometry

An ethyl chloroformate free fatty acid (ECF-FFA) method was developed for quantification of free fatty acids (FFAs) in milk. The method is based on gas chromatography mass spectrometry with ECF in-solution derivatisation and was optimised for the comprehensive analysis of FFAs (C₄–C_18:3) in bovine milk. Good estimations of all FFAs, including the most water-soluble short chain free fatty acids were achieved by using in-solution derivatisation, which can be ascribed to the fact that an extraction of the milk samples with an organic phase is avoided. FFA standard solutions and raw milk samples were used to validate the proposed method. Correlation coefficients of >0.999 were obtained for all of the standards. Both the repeatability and the within three days stability of the analytical method were high (RSD <4%) for the quantified FFAs in milk. Method recovery of spiked milk samples with three different levels of FFAs was in the range 94–103%.     

Enhancement and inhibition of denitrification by fluid-flow and dissolved oxygen flux to stream sediments

Microscale measurements of nitrate (NO3-) and dissolved oxygen (DO) concentrations in sediments were made in a laboratory channel under turbulent fluid-flow conditions to examine the effects of DO flux on denitrification rates. DO concentrations and flux within sediments increased with increasing velocity in the surface water. Under low fluid-flow conditions (shear stress velocity, u* < 0.23 cm s(-1)), increasing velocity increased NO3- loss from the bulk flow. For high fluid-flow conditions (u* > 0.39 cm s(-1)), increasing velocity inhibited NO3-loss. Sediment cores were collected and sliced to measure the depth distribution of denitrifying biomass in sediments. Quantities of nirK and nirS genes were higher within the surface layer and decreased with depth in the sediments. Microscale concentration profiles of DO and NO3- revealed that denitrification occurs within a thin region just below the oxic-anoxic interface in sediments. The interplay of mass transfer and DO flux generated threshold conditions for NO3- loss by denitrification. These results suggest that for a given sediment and environmental conditions (chemical, physical, microbiological), there exists an optimal range in velocities for enhancing denitrification in aquatic systems.     

Enhanced nitrate removal efficiency in wetland microcosms using an episediment layer for denitrification

Microbial denitrification is the principal removal mechanism for nitrogen in treatment wetlands. For this study, flow-through wetland microcosms were designed to testwhether variations in the macroporous structure of the denitrification zone affected overall nitrate removal. In a sediment-only treatment, carbon as cattail (Typha latifolia) litter was mixed throughout a porous sediment matrix. A second treatment contained a distinct layer of loosely aggregated litter pieces placed atop the sediment matrix to form an episediment zone. Results showed that across nine conditions (NO3- influent = 7, 36, 65 mg N L(-1) and t(res) = 2.2, 2.9, 4.4, 7.7 d; n = 48) average denitrification was 33% greater (p < 0.0001) in the episediment treatment than in the sediment-only treatment. Both sediment-only and episediment treatments followed apparent reaction kinetics close to first order, with k(epi) = 0.21 d(-1) for the episediment treatment and k(sed) = 0.12 d(-1) for the sediment-only treatment. Analysis of vertical nitrate profile data using diffusive and turbulent mixing models indicated that denitrification occurred in a partially mixed episediment layer, as well as in an underlying sediment layer. Approximately 40% of the nitrate removal that occurred in the episediment treatment was estimated to occur in the episediments themselves. We conclude that enhancementof an episediment layer can increase denitrification in treatment wetlands which receive nitrate in overlying water.     

New approach for the quantification of processed animal proteins in feed using light microscopy

A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.     

An integrative approach to identify Bacillus weihenstephanensis resistance biomarkers using gene expression quantification throughout acid inactivation

The aim of this study was to define an integrative approach to identify resistance biomarkers using gene expression quantification and mathematical modelling. Mid-exponentially growing cells were transferred into acid conditions (BHI, pH 4.6) to obtain inactivation kinetics, performed in triplicate. The inactivation curve was fitted with a mixed Weibull model. This model allowed to differentiate two subpopulations with various acid resistances among the initial population. In parallel, differential gene expression was quantified by RT-qPCR. While narL was down-regulated throughout acid inactivation, sigB and katA were up-regulated. sigB expression up-regulation peak was correlated to the less resistant subpopulation when katA up-regulation, was correlated to the more resistant subpopulation. Moreover, differences in population structure were highlighted between each replicate. The higher proportion of the more resistant subpopulation was linked to a higher katA gene expression. These results suggest that sigB and katA might be used as different types of biomarkers, for instance to track moderate and high acid-resistance, respectively. The use of this approach combining RT-qPCR and predictive modelling to track cellular biomarker variations appears as an interesting tool to take into account physiological cell responses into mathematical modelling, allowing an accurate prediction of microbial behaviour.     

A stable isotope approach for the quantification of sewer infiltration

Extraneous flows in wastewater collection systems are conventionally evaluated solely on the consideration of discharge hydrographs, which often involves a great degree of subjectivity and oversimplification. To obtain reliable information on the material fluxes within the urban environment, the use of intrinsic tracers can be the optimal choice. We demonstrate the successful use of naturally occurring stable isotopes of water (18O/16O and D/H) to accurately quantify extraneous discharge of groundwater in a combined sewer network. Fresh water supply from a distant hydrological regime provided usable isotopic separations between drinking water (proxy for real foul sewage) and local groundwater (proxy for sewer infiltration) of 1.8 per thousand in delta18O and 11.7 per thousand in 62H. Diurnal variation of wastewater isotopic composition reflected both the varying rates of foul sewage production and irregular dispersion effects in the pipe network. The latter suggest the existence of larger cumulative backwater zone volumes, which have not been attended to yet. Infiltrating groundwater contributed 39% (95% confidence interval = +/- 2.5%) of the total daily dry weather wastewater discharge. This paper discusses all relevant aspects for practical application of the method. It presents a comprehensive framework for uncertainty analysis and details on the detection and discrimination of possibly interfering effects.     

Surface photochemistry of pesticides: an approach using diffuse reflectance and chromatography techniques

The photochemistry of pesticides triadimenol and triadimefon was studied on cellulose and beta-cyclodextrin (beta-CD) in controlled and natural conditions, using diffuse reflectance techniques and chromatographic analysis. The photochemistry of triadimenol occurs from the chlorophenoxyl moiety, while the photodegradation of triadimefon also involves the carbonyl group. The formation of 4-chlorophenoxyl radical is one of the major reaction pathways for both pesticides and leads to 4-chlorophenol. Triadimenol also undergoes photooxidation and dechlorination, leading to triadimefon and dechlorinated triadimenol, respectively. The other main reaction process of triadimefon involves alpha-cleavage from the carbonyl group, leading to decarbonylated compounds. Triadimenol undergoes photodegradation at 254 nm but was found to be stable at 313 nm, while triadimefon degradates in both conditions. Both pesticides undergo photochemical decomposition under solar radiation, being the initial degradation of rate per unit area of triadimefon 1 order of magnitude higher than the observed for triadimenol in both supports. The degradation rates of the pesticides were somewhat lower in beta-CD than on cellulose. Photoproduct distribution of triadimenol and triadimefon is similar for the different irradiation conditions, indicating an intramolecular energy transfer from the chlorophenoxyl moiety to the carbonyl group in the latter pesticide.     

Spectroscopic quantification of bacteria using artificial neural networks

Fourier transform-infrared spectroscopy, in conjunction with artificial neural networks, has been used for identification and classification of selected foodborne pathogens. Five bacterial species (Enterococcus faecium, Salmonella Enteritidis, Bacillus cereus, Yersinia enterocolitica, Shigella boydii) and five Escherichia coli strains (O103, O55, O121, O30, O26) suspended in phosphate-buffered saline were enumerated to provide seven different concentrations ranging from 10(9) to 10(3) CFU/ ml. The trained artificial neural networks were then validated with an independent subset of samples and compared with the traditional plate count method. It was found that the concentration-based classification of the species was 100% correct and the strain-based classification was 90 to 100% accurate.     

Nanofiltration membrane fouling by oppositely charged macromolecules: investigation on flux behavior, foulant mass deposition, and solute rejection

Nanofiltration membrane fouling by oppositely charged polysaccharide (alginate) and protein (lysozyme) was systematically studied. It was found that membrane flux decline in the presence of both lysozyme and alginate was much more severe compared to that when there was only lysozyme or alginate in the feed solution. The flux performance for the mixed foulants was only weakly affected by solution pH and calcium concentration. These effects were likely due to the strong electrostatic attraction between the two oppositely charged foulants. Higher initial flux caused increased foulant deposition, more compact foulant layer, and more severe flux decline. The deposited foulant cake layer had a strong tendency to maintain a constant foulant composition that was independent of the membrane initial flux and only weakly dependent on the relative foulant concentration in feed solution. In contrast, solution chemistry (pH and [Ca2?]) had marked effect on the foulant layer composition, likely due to the resulting changes in the foulant-foulant interaction. The mixed alginate-lysozyme fouling could result in an initial enhancement in salt rejection. However, such initial enhancement was not observed when there was 1 mM calcium present in the feedwater, which may be attributed to the charge neutralization of the foulant layer.     

Correlation between isotopic and meteorological parameters in Italian wines: a local-scale approach

BACKGROUND: Since the beginning of the 1980s deuterium nuclear magnetic resonance and carbon‐13 mass spectrometry have proved to be reliable techniques for detecting adulteration and for classifying natural products by their geographic origin. Scientific literature has so far mainly focused on data acquired at regional level where isotopic parameters are correlated to climatic mean data relative to large territories. RESULTS: Nebbiolo and Barbera wine samples of various vintages and from different areas within the Piedmont region (northern Italy) were analysed using SNIF‐NMR and GC‐C‐IRMS and a large set of meteorological parameters were recorded by means of weather stations placed in fields where the grapes were grown. Correlations between isotopic (²H and ¹³C) data and several climatic parameters at a local level (mean temperature, total rainfall, mean humidity and thermal sums) were attempted and some linear correlations were found. CONCLUSIONS: Mean temperature and total rainfall were found to be correlated to isotopic (²H and ¹³C) abundance in linear direct and inverse proportions respectively. Lower or no correlations between deuterium and carbon‐13 abundances and other meteorological parameters such as mean humidity and thermal sums were found. Moreover, wines produced from different grape varieties in the same grape field showed significantly different isotopic values. Copyright © 2011 Society of Chemical Industry     

Modeling lead input and output in soils using lead isotopic geochemistry

The aim of this study is to model downward migration of lead from the plow layer of an experimental site located in Versailles (about 15 km southwest of Paris, France). Since 1928, samples have been collected annually from the topsoil of three control plots maintained in bare fallow. Thirty samples from 10 different years were analyzed for their lead and scandium contents and lead isotopic compositions. The fluxes are simple because of the well-controlled experimental conditions in Versailles: only one output flux, described as a first-order differential function of the anthropogenic lead pool, was taken into account; the inputs were exclusively ascribed to atmospheric deposition. The combination of concentration and isotopic data allows the rate of migration from the plowed topsoil to the underlying horizon and, to a lesser extent, the atmospheric fluxes to be assessed. Both results are in good agreement with the sparse data available. Indeed, the post-depositional migration of lead appears negligible at the human time scale: less than 0.1% of the potentially mobile lead pool migrates downward, out of the first 25 cm of the soil, each year. Assuming future lead inputs equal to 0, at least 700 yr would be required to halve the amount of accumulated lead pollution. Such a low migration rate is compatible with the persistence of a major anthropogenic lead pool deposited before 1928. Knowledge of pollution history seems therefore to be of primary importance.     

三酰甘油氧化物单体的HPSEC-GC/MS法高灵敏度定量测定

油脂在高温和氧气环境下形成非挥发性物质,主要为三酰甘油氧化物(ox–TG)。油脂中ox–TG的量与油脂的氧化程度呈现良好的线性关系。因此通过对ox–TG的精确定量可以客观地反映出油脂的氧化程度。鉴于传统分子排阻色谱法对ox–TG精确定量存在一定的缺陷,利用HPSEC–GC/MS技术,建立了一种用于食用植物油中三酰甘油氧化物单体(ox–TGM)高灵敏度的定量分析方法,具有良好的重现性和精度。结果表明,油脂中ox–TGM的定量限和检测限分别达到0.075、0.023 ppm。     

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.     

Rapid quantification of Escherichia coli O157:H7 in lettuce and beef using an on-chip staining microfluidic device

Quality assurance is one of the fundamental ways of preventing infections from foodborne pathogens such as Escherichia coli O157:H7, which produces a deadly toxin. Simple, rapid, and accurate methods for the detection of foodborne pathogens are necessary for healthcare management. In the present study, we applied our microfluidic device, which uses a fluorescent staining-based detection system, to enumerate E. coli O157:H7 cells in lettuce and beef samples. E. coli O157:H7 cells spiked into lettuce or beef samples were collected using a 0.2-μm-pore-sized filter or a two-step centrifugation process. The recovery ratios of inoculated E. coli O157:H7 cells from the lettuce and beef samples counted using fluorescence microscopy were 84 (± 10)% and 90 (± 7.3)%, respectively. The counts of E. coli O157:H7 inoculated into lettuce and beef obtained using the microfluidic system were close to the counts obtained using fluorescence microscopy. Our microfluidic approach offers a semi-automated platform for the quantitative detection of microbial cells from complex food samples and facilitates quantification of microbes in food and food production lines within 1 hr.     

PCR-based quantification of genetically modified Bt maize: single-competitive versus dual-competitive approach

 Methods for the quantification of transgenic insect-resistant Bt maize by single- and dual-competitive PCR were developed. The analysis of mixtures of DNA solutions, as well as of maize flours containing defined amounts of Bt maize, demonstrated the usefulness of single-competitive PCR based on coamplification of the CDPK promoter/cryIA(b) gene region of Bt maize and an internal standard. Upon heat treatment of DNA solutions and maize flour, respectively, the recovery of the Bt proportion compared to the starting material determined by single-competitive PCR decreased significantly. This systematic error could be compensated for by using a dual-competitive approach based on PCR quantification of the transgenic target sequence of Bt maize and of the maize specific invertase gene (ivr1). Received: 24 January 2000 / Revised version: 25 February 2000