Evaluation of the impact of buffered peptone water composition on the discrimination between Salmonella enterica and Escherichia coli by Raman spectroscopy.

The detection of Salmonella spp. in food samples is regulated by the ISO 6579:2002 standard, which requires that precise procedures are followed to ensure the reliability of the detection process. This standard requires buffered peptone water as a rich medium for the enrichment of bacteria. However, the effects of different brands of buffered peptone water on the identification of microorganisms by Raman spectroscopy are unknown. In this regard, our study evaluated the discrimination between two bacterial species, Salmonella enterica and Escherichia coli, inoculated and analyzed with six of the most commonly used buffered peptone water brands. The results showed that bacterial cells behaved differently according to the brand used in terms of biomass production and the spectral fingerprint. The identification accuracy of the analyzed strains was between 85% and 100% depending on the given brand. Several batches of two brands were studied to evaluate the classification rates between the analyzed bacterial species. The chemical analysis performed on these brands showed that the nutrient content was slightly different and probably explained the observed effects. On the basis of these results, Raman spectroscopy operators are encouraged to select an adequate culture medium and continue its use throughout the identification process to guarantee optimal recognition of the microorganism of interest.

Main Technological Advancements in Bacterial Bioluminescent Biosensors Over the Last Two Decades.

Environmental quality assessment is an extensive field of research due to the permanent increase of the stringency imposed by the legislative framework. To complete the wide panel of measurement methods, essentially based on physicochemical tools, some scientists focused on the development of alternative biological methods such as those based on the use of bioluminescent bacteria biosensors. The first report dedicated to the development of such biosensors dates back to 1967 and describes an analytical system designed to address the problem of air toxicity assessment. Nevertheless the available technologies in the photosensitive sensors field were not mature enough and, as a result, limited biosensor development possibilities. For about 20 years, the wide democratisation of photosensors coupled with advances in the genetic engineering field have allowed the expansion of the scope of possibilities of bioluminescent bacterial biosensors, allowing a significant emergence of these biotechnologies. This chapter retraces the history of the main technological evolutions that bacterial bioluminescent biosensors have known over the last two decades. Graphical Abstract.

Detection of Metal and Organometallic Compounds with Bioluminescent Bacterial Bioassays.

Chemical detection of metal and organometallic compounds is very specific and sensitive, but these techniques are time consuming and expensive. Although these techniques provide information about the concentrations of compounds, they fail to inform us about the toxicity of a sample. Because the toxic effects of metals and organometallic compounds are influenced by a multitude of environmental factors, such as pH, the presence of chelating agents, speciation, and organic matter, bioassays have been developed for ecotoxicological studies. Among these bioassays, recombinant luminescent bacteria have been developed over the past 20 years, and many of them are specific for the detection of metals and metalloids. These bioassays are simple to use, are inexpensive, and provide information on the bioavailable fraction of metals and organometals. Thus, they are an essential complementary tool for providing information beyond chemical analysis. In this chapter, we propose to investigate the detection of metals and organometallic compounds with bioluminescent bacterial bioassays and the applications of these bioassays to environmental samples. Graphical Abstract.

Potential of Raman Spectroscopy To Monitor Arsenic Toxicity on Bacteria: Insights toward Multiparametric Bioassays.

In the field of toxicological bioassays, the latest progress in Raman spectroscopy opens new research perspectives on a fast method of observing metabolic responses against toxic agents. This technique offers a multiparametric approach, providing an overview of the physiological changes that are caused by pollutants. However, physiological spectral fingerprints require complex chemometric methods for proper analysis. In this study, particular attention has been given to the elaboration of an "aberrant spectra" detection strategy to highlight the effects of arsenic on the bacteria Escherichia coli. This strategy significantly improved spectra classification, consistent with a dose-response effect of the four tested concentrations of the metal. Indeed, the correct classification score of the spectra increased from 88 to more than 99%. The exposure time effect has also been investigated. The fine analysis of Raman spectroscopy fingerprints enabled the design of different "spectral signatures", highlighting early and late effects of arsenic on bacteria. The observed variations are in agreement with the expected toxicity and encourage the use of Raman spectroscopy for toxic element detection.

Raman spectroscopy applied to the horizontal methods ISO 6579:2002 to identify Salmonella spp. in the food industry.

Food safety is a major concern for suppliers in the food chain to ensure the safety of their products. The identification procedure requested by norms is tedious, and it often requires systematic controls and qualified staff to perform the necessary analyses. Raman spectroscopy offers new opportunities to rapidly and efficiently ascertain the presence of pathogens in samples. Nevertheless, this technique requires a standardized procedure to be applied in the industrial context. Our study shows that the variability between spectral fingerprints is related to the physiological state of the microbial species and the growth phase of the bacteria plays a crucial role in its identification by Raman spectroscopy. To improve the discrimination between closely related bacterial species, a procedure based on the selection of bacterial spectra in the exponential growth phase was proposed. Different ways to introduce Raman spectroscopy in the ISO 6579:2002 standards are also proposed from the entire process to a shorter protocol. In the latter case, the identification of bacterial colonies after the selective enrichment step was proposed with the advantages of this path in terms of simplicity and rapidity (analysis time is reduced up to 50 h from the 100 h required by the standard). The protocol validated using six food categories from industrial partners have presented a good correlation by confirmation with other laboratory classical methods. In the future, this procedure could be introduced to the control system of the food production chain with a reliable database for various microorganisms encountered in this field.

Methods for assessing biochemical oxygen demand (BOD): a review.

The Biochemical Oxygen Demand (BOD) is one of the most widely used criteria for water quality assessment. It provides information about the ready biodegradable fraction of the organic load in water. However, this analytical method is time-consuming (generally 5 days, BOD5), and the results may vary according to the laboratory (20%), primarily due to fluctuations in the microbial diversity of the inoculum used. Work performed during the two last decades has resulted in several technologies that are less time-consuming and more reliable. This review is devoted to the analysis of the technical features of the principal methods described in the literature in order to compare their performances (measuring window, reliability, robustness) and to identify the pros and the cons of each method.

New insights into polyurethane biodegradation and realistic prospects for the development of a sustainable waste recycling process.

Polyurethanes are polymeric plastics that were first used as substitutes for traditional polymers suspected to release volatile organic hazardous substances. The limitless conformations and formulations of polyurethanes enabled their use in a wide variety of applications. Because approximately 10 Mt of polyurethanes is produced each year, environmental concern over their considerable contribution to landfill waste accumulation appeared in the 1990s. To date, no recycling processes allow for the efficient reuse of polyurethane waste due to their high resistance to (a)biotic disturbances. To find alternatives to systematic accumulation or incineration of polyurethanes, a bibliographic analysis was performed on major scientific advances in the polyurethane (bio)degradation field to identify opportunities for the development of new technologies to recondition this material. Until polymers exhibiting oxo- or hydro-biodegradative traits are generated, conventional polyurethanes that are known to be only slightly biodegradable are of great concern. The research focused on polyurethane biodegradation highlights recent attempts to reprocess conventional industrial polyurethanes via microbial or enzymatic degradation. This review describes several wonderful opportunities for the establishment of new processes for polyurethane recycling. Meeting these new challenges could lead to the development of sustainable management processes involving polymer recycling or reuse as environmentally safe options for industries. The ability to upgrade polyurethane wastes to chemical compounds with a higher added value would be especially attractive.

Polythiophene synthesis coupled to quartz crystal microbalance and Raman spectroscopy for detecting bacteria.

A simple electrochemical procedure was used for the synthesis of a polythiophene containing para-benzenesulfonyl chloride groups. The obtained polymer was shown to be very reactive and directly able to covalently bind nucleophile biomolecules. Protein A and a specific antibody were then successively immobilized on the conductive polymer through a covalent bonding of Protein A with the as-prepared linker for bacteria trapping purpose. All reactions were controlled in situ by cyclic voltammetry, quartz crystal microbalance and Raman spectroscopy. The results were compared to those previously obtained on gold surface modified with the same chemical linker. The conductive polymer led to a very high rate of antibody recognition compared to the gold surface and to literature, probably due to a large available surface obtained after polymerization. One example of pathogenic bacteria "Salmonella enterica paratyphi" detection was successfully tested on the substrates. The presented results are promising for the future design of simple and inexpensive immunocapture-based sensors.

A simple method of surface functionalisation for immuno-specific immobilisation of proteins.

We present a new and advanced methodology, developed for surface functionalisation of gold and to study immobilisation of an immuno-specific system of proteins. A combination of electrochemical quartz crystal microbalance and Raman spectroscopy techniques allowed a complete understanding of the system starting from surface functionalisation and progressing to the functional structure analysis of immobilised proteins. A simple electrochemical procedure was formulated to prepare sulphonyl chloride terminated gold surfaces that form a strong sulphonamide bond with the receptor protein staphylococcal protein A (SpA). On the SpA grafted surfaces, the immobilisation of a human IgG and consecutive binding of an immuno-specific anti-human IgG was observed. The surface functional groups form a strong interaction with SpA without disturbing its functional properties. The native functional structure of SpA and also the IgGs was found to be retained in their immobilised state.

Optimization and validation of a simple method using P22::luxAB bacteriophage for rapid detection of Salmonella enterica serotypes A, B, and D in poultry samples.

A simple method was developed for the fast and inexpensive detection of Salmonella Typhimurium using a recombinant P22::luxAB phage. All the steps from phage production to detection were considered. A strain of Salmonella Typhimurium harboring the prophage P22::luxAB was grown in batch culture to produce spontaneously the recombinant bacteriophage. Batch production to stationary phase was better for propagation of the phage and led to a total population of 4.3 x 10(9) (+/-4.3 x 10(9)) PFU/ml of P22, including only 1.4 x 10(6) (+/-1 x 10(6)) PFU/ml harboring the luxAB genes. After preenrichment, a simple four-step bioassay was tested and optimized for several parameters. The detection limit of the luminometer was only 5 x 10(2) (+/-1.75 x 10(2)) CFU Salmonella Typhimurium per ml, but increased to 1.5 x 10(4) (+/-1.17 x 10(4)) CFU Salmonella Typhimurium per ml when the cells were in a complex matrix. The detection limit after the preenrichment was 6.5 x 10(3) (+/-1.5 x 10(3)) CFU Salmonella Typhimurium per ml, but the detection limit after the preenrichment also increased markedly to 1.65 x 10(5) (+/-0.15 x 10(5)) CFU Salmonella Typhimurium per ml when Salmonella Typhimurium was in a complex matrix. Finally, the bioassay was applied to the detection of Salmonella Typhimurium LT2 in 14 different feed and environmental samples (including duck feed, litters, and feces) spiked either before or after the preenrichment process. It was possible to detect Salmonella Typhimurium LT2 in all samples within 16 h.

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain.

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.

Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand.

The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd3+). The uptake by sand of this element was also measured. Saturation curves and Scatchard models were established for all biosorbants used in this work. The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd3+, which ranged from 350 micromol g(-1) for B. subtilis to 5.1 micromol g(-1) for S. cerevisiae. This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations. Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells. Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension.

Laboratory evaluation of crude oil biodegradation with commercial or natural microbial inocula.

Experiments have been performed to screen eight microbial commercial products that, according to the manufacturers, are able to degrade crude oil. This study compared the crude oil biodegradation activity of commercial inocula with that of natural inocula (activated sludge and tropical aquarium water). Some of the latter were previously adapted to the crude oil as the only carbon source. Nutrients and sorbents in the commercial formulations were eliminated, and each inoculum was precultured on marine yeast extract medium. Crude oil biodegradability tests were conducted with close initial substrate concentration to initial bacterial concentration ratios (S0/X0) of 0.94 g of crude oil/10(9) CFU, which allowed a comparison of biodegradation activity. The inocula oxidized the crude oil after a short lag time of less than 3-18 days. After that time, the rate of oxidation varied between 45 and 244 mg O2/(L.day). Crude oil biodegradation after a 28-day test was effective only for 10 out of 12 inocula (from 0.1 to 25% in weight). Biodegradation mainly corresponded to the saturated fraction of the crude oil; the asphaltene fraction was never significantly biodegraded. Our results led to the conclusion that natural inocula, either adapted or not adapted to crude oil, were the most active (from 16 to 25% of loss in crude oil weight) and only one commercial inoculum was able to degrade 18% of the crude oil. Other inocula had a biodegradation activity ranging from 0.1 to 14%.

Preadapted inocula for limiting the risk of errors in biodegradability tests.

Reducing the time for biodegradability tests to 28 days poses a problem when the inoculum contains few biodegraders, as a biodegradable xenobiotic must give a positive result within this time. The influence of initial concentration (X0, number of cells liter-1) on the lag time (hours) of para-nitrophenol biodegradability tests was examined using different concentrations of adapted Pseudomonas putida with para-nitrophenol as the sole carbon and energy source. Lag time decreased as bacterial density increased according to the expression y = 63.5 - 5.7(log10X0). The addition of river water to the P. putida concentrations shortened the lag time-bacterial density relationship and lag time filled the expression y = 52.4 - 5.1(log10X0). The addition of river water also increased the rate of para-nitrophenol biodegradation from 1.29 mgC (liters x hr)-1 to 2.11 mgC (liters x hr)-1. An examination of the effect of the initial adapted P. putida density, expressed as total cell, cultivable bacteria, or biodegraders, was also made on the para-nitrophenol biodegradability test outcome. The model-related cell density and the probability of test response give very similar k constants (kT = 0.56 x 10(-3) liter total cells-1; kv = 0.11 x 10(-3) liter CFU-1, kMPN = 0.16 x 10(-3) liter MPN-1). Comparisons with nonadapted natural mixed culture (activated sludge, river water) indicate that the biodegradability test responses were the same as with adapted cells when the nonadapted cell concentrations were at least 5 x 10(10) total cells liter-1. As this high cell concentration led to carbon contamination, adapting mixed inocula before the test to increase the number of biodegraders appears to be the best solution. Before biodegradability tests, cell density can be adjusted using techniques which are not specific to biodegraders, and only 10(5) total adapted cells liter-1 are needed for a 99.9% chance of a positive response in para-nitrophenol biodegradability tests.

Bacterial inoculum density and probability of para-nitrophenol biodegradability test response.

This study has been carried out to establish a model linking probability of positive response in para-nitrophenol biodegradability test to controlled variables of the test (suspended solids, SS; total bacteria, AODC; cultivable bacteria, CFU; specific biodegraders, MPN). Series of dilution of 11 raw inocula (6 activated sludges, 5 river waters) were tested. They reveal very dispersed values of biomass measured as SS, AODC, and CFU and quite comparable values of specific biodegraders for each category of inoculum (river or sludge). The proposed model fits well the empirical distribution of the experimental frequency of positive results versus inoculum density for each controlled variable. The constants k of the model, representing the fraction of biodegraders for each inoculum, were tested by the likelihood ratio test and were proven to be different from one another according to the biomass descriptor and the origin of the inoculum. The probabilistic model, in the case of para-nitrophenol biodegradation, indicates that standardized official tests (closed bottle, AFNOR, Sturm, and MITI I) are seldom optimal under those conditions. It allows the determination of which inoculum concentration can lead to a high probability (e.g., 99.9%) of observing paranitrophenol biodegradation by raw inocula.