Fabienne Quilès

Infrared and Raman spectroscopical studies of salicylic and salicylate derivatives in aqueous solution

The assignment of vibrations of salicylic acid, salicylate monoanion and salicylate bianion in aqueous solution are discussed with the help of Raman spectroscopy, of attenuated total reflection infrared spectroscopy and of the computational density functional theory (DFT) approach.

In Situ Monitoring of the Nascent Pseudomonas fluorescens Biofilm Response to Variations in the Dissolved Organic Carbon Level in Low-Nutrient Water by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy

ABSTRACT Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent Pseudomonas fluorescens biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent P. fluorescens biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.

Hydrolysis of Uranyl(VI) in Acidic and Basic Aqueous Solutions Using a Noncomplexing Organic Base: A Multivariate Spectroscopic and Statistical Study

In the field of actinide aqueous chemistry, this work aims to resolve some controversy about uranyl(VI) hydroxide species present in basic aqueous solutions. We revisit the Raman, IR, and UV-visible spectra with two new approaches. First, Raman, IR and UV data were recorded systematically from aqueous solutions with the noncomplexing electrolyte (C(2)H(5))(4)NNO(3) at 25 °C and 0.1 MPa ([U(total)] = 0.005-0.105 M) in H(2)O and D(2)O over a wide range of -log mH(D)(+) between 2.92 and 14.50. Second, vibrational spectra (IR and Raman) of basic solutions in H(2)O and D(2)O were analyzed using the Bayesian Positive Source Separation method to estimate pure spectra of individual species. In D(2)O solutions, the new spectroscopic data showed the occurrence of the same species as those in H(2)O. As observed for the wavenumber of the symmetric stretching mode, the wavenumber characteristic of the O═U═O antisymmetric stretching mode decreases as the number of OH(D)(-) ligands increases. These kinds of data, completed by (1) analysis of the signal widths, (2) persistence of the apparent exclusion rule between IR and Raman spectra of the uranyl species stretching modes, and (3) interpretation of the absorption UV-visible spectra, allow discussion of the chemistry, structures, and polynuclearity of uranyl(VI) species. In moderate basic solutions, the presence of two trimers is suggested. In highly basic solutions ([OH(-)] ≈ 3 M), the two monomers UO(2)(OH)(4)(2-) and UO(2)(OH)(5)(3-) are confirmed to be in good agreement with earlier EXAFS and NMR results. The occurrence of the UO(2)(OH)(6)(4-) monomer is also suggested from the more basic solutions investigated.

Analysis of changes in attenuated total reflection FTIR fingerprints of Pseudomonas fluorescens from planktonic state to nascent biofilm state.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is a useful method for monitoring biofilm in situ, non-destructively, in real time, and under fully hydrated conditions. In this work we focused on changes in Pseudomonas fluorescens ATR-FTIR fingerprint accompanying the very early stages of biofilm formation: initial bacterial adhesion and the very beginning of biofilm development in the presence of nutrients. To help interpreting variations in the ATR-FTIR fingerprint of sessile bacteria, ATR-FTIR spectra of planktonic bacteria in different growth phases were also examined, and the average surface coverage and spatial arrangement of bacteria on the ATR crystal were determined by epifluorescence microscopy. The proteins, nucleic acids and polysaccharides ATR-FTIR spectral data recorded during growth of sessile bacteria were shown to be linked to changes in the physiological state of the bacteria, possibly accompanied by extracellular polymeric substances production. This work clearly showed by spectroscopic method how bacteria change drastically their metabolism during the first hours of biofilm formation.

Infrared and Raman spectroscopic study of carboxylic acids in heavy water

Infrared and Raman spectra of CH3COOD and CD3COOD were obtained below 1900 cm−1, from dilute solutions in heavy water up to neat liquids. The relation of the intermolecular interactions with the acid concentration is discussed. In the debate concerning the structure of pure liquid acetic acid at room temperature, we favour the assignment of the major species to the centrosymmetric dimer. This is based not only on the literature data for crystal, liquid and vapour acetic acid, but also on the infrared and Raman spectra of liquid acetic and propanoic acids and their 0.1 M solutions in water. On increasing the acid concentration from dilute solutions, the hydrated monomer is progressively replaced by more or less hydrated linear dimer and then by the cyclic dimer. At a molar fraction of 0.5, the three kinds of species coexist. At still higher concentrations, longer oligomers appear while the cyclic dimer dominates the other species. A comprehensive assignment of the infrared and Raman spectra is given on the basis of a previously published ab initio calculation for monomer, cyclic and linear dimers. A narrow well resolved satellite band is observed for the hydrated monomer some 45 cm−1 above the CC bond stretching vibration (νCC), not only for CH3COOD and CD3COOD but also for C2H5COOD and CH2ClCOOD, specifically in dilute heavy water solutions. It is not easily assigned to overtones or combination bands simultaneously for the three molecules. Both a blue-shift and a narrowing of the νCC band are usually observed by ionisation of a carboxylic acid into a carboxylate ion in water. The hypothesis of a contact ion pair {R–COO−, D3O+}, whose protonated equivalent species would not exist in normal water, is thus discussed.

Ab initio study of the vibrational properties of acetic acid monomers and dimers

The vibrational spectra of CH3COOH, CH3COOD, CD3COOH and CD3COOD, either monomeric or in two dimeric structures (centrosymmetric or side-on linear, with two bent hydrogen bonds), were studied by ab initio calculations at the DFT/B3LYP/6-31++G** level. The force field, the potential energy distribution and the derivatives of the electric dipole moment with respect to internal coordinates were analysed in addition to the vibration wavenumbers. A comprehensive discussion of the infrared intensities between 1150 and 1500 cm−1 is made for the twelve structures studied. The calculated infrared intensities of monomers are in close agreement with the experimental spectra. In particular, the puzzling wavenumber increase that had been observed in argon matrices for the intense band assigned to the COH angle bending δCOH, by substituting OD for OH, is explained without invoking the previous hypothesis of the tunnelling of the H atom between the two O atoms. It is related to the decoupling of δCOH from a mode involving the stretching of the C–C and C–O bonds with opposite phases.

Durable cofactor immobilization in sol-gel bio-composite thin films for reagentless biosensors and bioreactors using dehydrogenases.

A new strategy directed to the durable immobilization of NAD(+)/NADH cofactors has been tested, along with a suitable redox mediator (ferrocene), in biocompatible sol-gel matrices encapsulating a bi-enzymatic system (a dehydrogenase and a diaphorase, this latter being useful to the safe regeneration of the cofactor), which were deposited as thin films onto glassy carbon electrode surfaces. It involves the chemical attachment of NAD(+) to the silica matrix using glycidoxypropylsilane in the course of the sol-gel process (in smooth chemical conditions). This approach based on chemical bonding of the cofactor (which was checked by infrared spectroscopy) led to good performances in terms of long-term stability of the electrochemical response. The possibility to integrate all components (proteins, cofactor, mediator) in the sol-gel layer in an active and durable form gave rise to reagentless devices with extended operational stability (i.e. high amperometric response maintained for more than 12h of continuous use under constant potential, whereas the signal completely vanished within the first few minutes of working with non-covalently bonded NAD(+)). To confirm the wide applicability of the proposed approach, the same strategy has been applied to the elaboration of biosensors for D-sorbitol, D-glucose and L-lactate with using D-sorbitol dehydrogenase, D-glucose dehydrogenase and L-lactate dehydrogenase respectively. The analytical characteristics of the glucose sensors are given and compared to previous approaches described in the literature for the elaboration of reagentless biosensors.

Production of extracellular glycogen by Pseudomonas fluorescens: spectroscopic evidence and conformational analysis by biomolecular recognition.

Glycogen is mainly found as the principal storage form of glucose in cells. Many bacteria are able to synthesize large amounts of glycogen under unfavorable life conditions. By combining infrared spectroscopy, single molecule force spectroscopy (SMFS) and immuno-staining technique, we evidenced that planktonic P. fluorescens (Pf) cells are also able to produce glycogen as an extracellular polymeric substance. For this purpose, Pf suspensions were examined at 3 and 21 h of growth in nutritive medium (LB, 0.5 g/L). The conformation of the extracellular glycogen, revealed through its infrared spectral signature, has been investigated by SMFS measurements using Freely Jointed Chain model. The analysis of force versus distance curves showed over growth time that the increase of glycogen production was accompanied by an increase in glycogen contour lengths and ramifications. These results demonstrated that the production of extracellular bacterial glycogen can occur even if the cells are not subjected to unfavorable life conditions.

Monitoring the effect of organic matter on biofilm growth in low nutritive waters by ATR/FT-IR spectroscopy

Bacterial biofilm growth was followed in low nutritive media by the use of attenuated total reflectance Fourier transform infrared (ATR/FT-IR) spectroscopy, an in situ non-destructive method. The aims were to determine the effect of organic matter of different origins on the development of the biofilm and to evaluate the usefulness of the method as an early warning signal for changes in organic matter in drinking water. The assays were carried out with a strain of Enterobacter cloacae attached to a zinc selenide crystal, which is compatible with vibrational spectroscopic analysis. The E. cloacae biofilm was grown in flow cells and was continuously fed with various filtered (0.2 w m pore size) water samples. The water samples (drinking water, diluted river water, and diluted algal bloom) all contained the same dissolved organic carbon (DOC) concentrations (1.5-1.7 mg lm 1) but variable quantities of biodegradable organic matter. The development of biofilms was continuously monitored for 2 weeks at 20°C. Protein amide II and polysaccharides C-O and C-O-C stretching band areas were measured around 1550 cmm 1 and 1050 cmm 1, respectively. The results indicated that ATR/FT-IR spectroscopy could discriminate between the time courses of biofilms in the three water samples tested, especially protein signals, even after relatively short periods of time (10 h). E. cloacae biofilms developed well with algal bloom and river water but not with drinking water. The absorption signals obtained within the first 10 h could lead to the development of an early warning system for changes in the nutritive status of drinking water.

In situ and real time investigation of the evolution of a Pseudomonas fluorescens nascent biofilm in the presence of an antimicrobial peptide.

Against the increase of bacterial resistance to traditional antibiotics, antimicrobial peptides (AMP) are considered as promising alternatives. Bacterial biofilms are more resistant to antibiotics that their planktonic counterpart. The purpose of this study was to investigate the action of an AMP against a nascent bacterial biofilm. The activity of dermaseptin S4 derivative S4(1-16)M4Ka against 6 h-old Pseudomonas fluorescens biofilms was assessed by using a combination of Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy in situ and in real time, fluorescence microscopy using the Baclight™ kit, and Atomic Force Microscopy (AFM, imaging and force spectroscopy). After exposure to the peptide at three concentrations, different dramatic and fast changes over time were observed in the ATR-FTIR fingerprints reflecting a concentration-dependent action of the AMP. The ATR-FTIR spectra revealed major biochemical and physiological changes, adsorption/accumulation of the AMP on the bacteria, loss of membrane lipids, bacterial detachment, bacterial regrowth, or inhibition of biofilm growth. AFM allowed estimating at the nanoscale the effect of the AMP on the nanomechanical properties of the sessile bacteria. The bacterial membrane elasticity data measured by force spectroscopy were consistent with ATR-FTIR spectra, and they allowed suggesting a mechanism of action of this AMP on sessile P. fluorescens. The combination of these three techniques is a powerful tool for in situ and in real time monitoring the activity of AMPs against bacteria in a biofilm.