Florence Lagarde

Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment.

Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant. Global cycling of arsenic is affected by microorganisms. This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As[III]) into arsenate (As[V]) in liquid medium. The rate of the transformation depends on the cell density. Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The strain also exhibits high minimum inhibitory concentrations (MICs) for As[III] (6.65 mM (500 mg L-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L-1)) or lead (1.20 mM (250 mg L-1)). Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod. The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites.

Degradability of superparamagnetic nanoparticles in a model of intracellular environment: follow-up of magnetic, structural and chemical properties

The unique magnetic properties of iron oxide nanoparticles have paved the way for various biomedical applications, such as magnetic resonance cellular imaging or magnetically induced therapeutic hyperthermia. Living cells interact with nanoparticles by internalizing them within intracellular acidic compartments. Although no acute toxicity of iron oxide nanoparticles has been reported up to now, the mechanisms of nanoparticle degradation by the cellular environment are still unknown. In the organism, the long term integrity and physical state of iron-based nanoparticles are challenged by iron homeostasis. In this study, we monitored the degradation of 7 nm sized maghemite nanoparticles in a medium mimicking the intracellular environment. Magnetic nanoparticles with three distinct surface coatings, currently evaluated as MRI contrast agents, were shown to exhibit different kinetics of dissolution at an acidic pH in the presence of a citrate chelating agent. Our assessment of the physical state of the nanoparticles during degradation revealed that the magnetic properties, size distribution and structure of the remaining nanocrystals were identical to those of the initial suspension. This result suggests a model for nanoparticle degradation with rapidly dissolved nanocrystals and a reservoir of intact nanoparticles.

Application of a sequential extraction procedure to study the release of elements from municipal solid waste incineration bottom ash

The release of five elements (Cr, Cu, Mn, Pb and Zn) from a municipal solid waste incineration bottom ash (BA) under different extraction conditions has been investigated by performing the three-step sequential extraction procedure proposed by the Standards, Measurements and Testing Program of the European Union. A fourth step (strong acid attack) has been added in order to calculate the mass balance. The results of this study provide information on the potential mobility of the studied elements. Almost all of the Cr and part of the Mn are extracted with strong acid which indicates low potential mobility in the environment. Most of the Cu is extracted under oxidizing conditions. Pb and Zn are released under acidic condition, indicating the possibility of their mobilization by changes in pH. The reproducibility of the sequential extraction procedure is also discussed.

Cell-based electrochemical biosensors for water quality assessment

During recent decades, extensive industrialisation and farming associated with improper waste management policies have led to the release of a wide range of toxic compounds into aquatic ecosystems, causing a rapid decrease of world freshwater resources and thus requiring urgent implementation of suitable legislation to define water remediation and protection strategies. In Europe, the Water Framework Directive aims to restore good qualitative and quantitative status to all water bodies by 2015. To achieve that, extensive monitoring programmes will be required, calling for rapid, reliable and cost-effective analytical methods for monitoring and toxicological impact assessment of water pollutants. In this context, whole cell biosensors appear as excellent alternatives to or techniques complementary to conventional chemical methods. Cells are easy to cultivate and manipulate, host many enzymes able to catalyse a wide range of biological reactions and can be coupled to various types of transducers. In addition, they are able to provide information about the bioavailability and the toxicity of the pollutants towards eukaryotic or prokaryotic cells. In this article, we present an overview of the use of whole cells, mainly bacteria, yeasts and algae, as sensing elements in electrochemical biosensors with respect to their practical applications in water quality monitoring, with particular emphasis on new trends and future perspectives. In contrast to optical detection, electrochemical transduction is not sensitive to light, can be used for analysis of turbid samples and does not require labelling. In some cases, it is also possible to achieve higher selectivities, even without cell modification, by operating at specific potentials where interferences are limited.

Determination of arsenic species in marine organisms by HPLC-ICP-OES and HPLC-HG-QFAAS

Separation and quantification of six arsenic species have been performed in cod, tuna and mussel samples by high performance liquid chromatography (HPLC) using inductively coupled plasma-optical emission spectrometry (ICP-OES) and hydride generation-quartz furnace atomic absorption spectrometry (HG-QFAAS) as detection techniques. It has been shown that arsenic extraction with a water-methanol (1∶1) mixture is sufficiently quantitative for the cod and tuna, in which arsenic is mainly present as arsenobetaine (about 90% of total As extracted). In contrast, only 60% of the element is extracted from the mussels and the chromatograms obtained reveal the presence of an unknown compound. Detection limits are in the μg ml−1 range for the HPLC-ICP-OES technique (quantification of arsenobetaine and arsenocholine) and in the ng ml−1 range for the HPLC-HG-QFAAS system (quantification of arsenite, arsenate, monomethylarsonic and dimethylarsinic acids).

Ultra-sensitive conductometric detection of heavy metals based on inhibition of alkaline phosphatase activity from Arthrospira platensis

This study is based on the conductometric measurement of alkaline phosphatase activity (APA) from the cyanobacterium, Arthrospira platensis, called Spirulina. Cyanobacterium cells were directly immobilized, by physical adsorption, on the ceramic part of gold interdigitated transducers. This activity was inhibited in the presence of heavy metals and a variation of the local conductivity was measured after addition of the substrate. The Michaelis-Menten constant (Km) was evaluated to be 0.75 mM through a calibration curve of the substrate, disodium 4-nitrophenylphosphate p-nitrophenyl phosphate (pNPP). Inhibition of APA was observed with cadmium and mercury with a detection limit of 10(-20) M. The half maximal inhibitory concentration (IC50) was determined at 10(-19) M for Cd(2+) and 10(-17) M for Hg(2+), and the binding affinity of heavy metal (Ki) was equal to the IC50. On the sensor surface, scanning electron microscopy (SEM) images revealed a remarkable evolution of the cyanobacteriums external surface that was attributable to the first defense mechanism against toxic heavy metals in trace. This effect was also confirmed through the important increase of response time τ(90%) recorded for APA response towards the substrate pNPP after cell exposure to metallic cations. Lifetime of the Spirulina-based biosensor was estimated to be more than 25 days.

RcnB Is a Periplasmic Protein Essential for Maintaining Intracellular Ni and Co Concentrations in Escherichia coli

Nickel and cobalt are both essential trace elements that are toxic when present in excess. The main resistance mechanism that bacteria use to overcome this toxicity is the efflux of these cations out of the cytoplasm. RND (resistance-nodulation-cell division)- and MFS (major facilitator superfamily)-type efflux systems are known to export either nickel or cobalt. The RcnA efflux pump, which belongs to a unique family, is responsible for the detoxification of Ni and Co in Escherichia coli. In this work, the role of the gene yohN, which is located downstream of rcnA, is investigated. yohN is cotranscribed with rcnA, and its expression is induced by Ni and Co. Surprisingly, in contrast to the effect of deleting rcnA, deletion of yohN conferred enhanced resistance to Ni and Co in E. coli, accompanied by decreased metal accumulation. We show that YohN is localized to the periplasm and does not bind Ni or Co ions directly. Physiological and genetic experiments demonstrate that YohN is not involved in Ni import. YohN is conserved among proteobacteria and belongs to a new family of proteins; consequently, yohN has been renamed rcnB. We show that the enhanced resistance of rcnB mutants to Ni and Co and their decreased Ni and Co intracellular accumulation are linked to the greater efflux of these ions in the absence of rcnB. Taken together, these results suggest that RcnB is required to maintain metal ion homeostasis, in conjunction with the efflux pump RcnA, presumably by modulating RcnA-mediated export of Ni and Co to avoid excess efflux of Ni and Co ions via an unknown novel mechanism.

Impedimetric immunosensor based on SWCNT-COOH modified gold microelectrodes for label-free detection of deep venous thrombosis biomarker

Measurement of D-dimer has subsequently become an essential element in the diagnostics of deep vein thrombosis and pulmonary embolism; in this context microelectrodes with an area of 9×10(-4) cm(2) were used to develop impedimetric immunosensor for detecting deep venous thrombosis biomarker (D-dimer). The biosensor is based on functionalized carbon nanotubes (SWCNT-COOH) where the antibody (anti-D-dimer) was immobilized by covalent binding. The electrical properties and the morphology of the biolayer were characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry and atomic force spectroscopy (AFM). Impedimetric microimmunosensor allows to obtain sensitivity of 40.1 kΩ μM(-1) and detection limit of 0.1 pg/mL (0.53 fM) with linear range from 0.1 pg/mL to 2 μg/mL (0.53 fM to 0.01 μM). We demonstrate that using carbon nanotubes and microelectrodes, high sensitivity and dynamic range were obtained. The biosensor exhibited a short response time of 10 min. Moreover, the studied immunosensor exhibits good reproducibility (R.S.D. 8.2%, n=4).

A rapid and sensitive alcohol oxidase/catalase conductometric biosensor for alcohol determination

A new conductometric biosensor has been developed for the determination of short chain primary aliphatic alcohols. The biosensor assembly was prepared through immobilization of alcohol oxidase from Hansenula sp. and bovine liver catalase in a photoreticulated poly(vinyl alcohol) membrane at the surface of interdigitated microelectrodes. The local conductivity increased rapidly after alcohol addition, reaching steady-state within 10 min. The sensitivity was maximal for methanol (0.394+/-0.004 microS microM(-1), n=5) and decreased by increasing the alcohol chain length. The response was linear up to 75 microM for methanol, 70 microM for ethanol and 65 microM for 1-propanol and limits of detection were 0.5 microM, 1 microM and 3 microM, respectively (S/N=3). No significant loss of the enzyme activities was observed after 3 months of storage at 4 degrees C in a 20mM phosphate buffer solution pH 7.2 (two or three measurements per week). After 4 months, 95% of the initial signal still remained. The biosensor response to ethanol was not significantly affected by acetic, lactic, ascorbic, malic, oxalic, citric, tartaric acids or glucose. The bi-enzymatic sensor was successfully applied to the determination of ethanol in different alcoholic beverages.

Heat-treated Saccharomyces cerevisiae for antimony speciation and antimony(III) preconcentration in water samples.

An analytical method was developed for antimony speciation and antimony(III) preconcentration in water samples. The method is based on the selective retention of Sb(III) by modified Saccharomyces cerevisiae in the presence of Sb(V). Heat, caustic and solvent pretreatments of the biomass were investigated to improve the kinetics and thermodynamics of Sb(III) uptake process at room temperature. Heating for 30 min at 80 degrees C was defined as the optimal treatment. Antimony accumulation by the cells was independent of pH (5-10) and ionic strength (0.01-0.1 mol L(-1)). 140 mg of yeast and 2h of contact were necessary to ensure quantitative sequestration of Sb(III) up to 750 microg L(-1). In these conditions, Sb(V) was not retained. Sb(V) was quantified in sorption supernatant by inductively coupled plasma mass spectrometry (ICP-MS) or inductively coupled plasma optical emission spectrometry (ICP-OES). Sb(III) was determined after elution with 40 mmol L(-1) thioglycolic acid at pH 10. A preconcentration factor close to nine was achieved for Sb(III) when 100mL of sample was processed. After preconcentration, the detection limits for Sb(III) and Sb(V) were 2 and 5 ng L(-1), respectively, using ICP-MS, 7 and 0.9 microg L(-1) using ICP-OES. The proposed method was successfully applied to the determination of Sb(III) and Sb(V) in spiked river and mineral water samples. The relative standard deviations (n=3) were in the 2-5% range at the tenth microg L(-1) level and less than 10% at the lowest Sb(III) and Sb(V) tested concentration (0.1 microg L(-1)). Corrected recoveries were in all cases close to 100%.