New Concepts in the Evaluation of Biodegradation/Persistence of Chemical Substances Using a Microbial Inoculum

The European REACH Regulation (Registration, Evaluation, Authorization of CHemical substances) implies, among other things, the evaluation of the biodegradability of chemical substances produced by industry. A large set of test methods is available including detailed information on the appropriate conditions for testing. However, the inoculum used for these tests constitutes a “black box.” If biodegradation is achievable from the growth of a small group of specific microbial species with the substance as the only carbon source, the result of the test depends largely on the cell density of this group at “time zero.” If these species are relatively rare in an inoculum that is normally used, the likelihood of inoculating a test with sufficient specific cells becomes a matter of probability. Normally this probability increases with total cell density and with the diversity of species in the inoculum. Furthermore the history of the inoculum, e.g., a possible pre-exposure to the test substance or similar substances will have a significant influence on the probability. A high probability can be expected for substances that are widely used and regularly released into the environment, whereas a low probability can be expected for new xenobiotic substances that have not yet been released into the environment. Be that as it may, once the inoculum sample contains sufficient specific degraders, the performance of the biodegradation will follow a typical S shaped growth curve which depends on the specific growth rate under laboratory conditions, the so called F/M ratio (ratio between food and biomass) and the more or less toxic recalcitrant, but possible, metabolites. Normally regulators require the evaluation of the growth curve using a simple approach such as half-time. Unfortunately probability and biodegradation half-time are very often confused. As the half-time values reflect laboratory conditions which are quite different from environmental conditions (after a substance is released), these values should not be used to quantify and predict environmental behavior. The probability value could be of much greater benefit for predictions under realistic conditions. The main issue in the evaluation of probability is that the result is not based on a single inoculum from an environmental sample, but on a variety of samples. These samples can be representative of regional or local areas, climate regions, water types, and history, e.g., pristine or polluted. The above concept has provided us with a new approach, namely “Probabio.” With this approach, persistence is not only regarded as a simple intrinsic property of a substance, but also as the capability of various environmental samples to degrade a substance under realistic exposure conditions and F/M ratio

Bacterial Bioluminescent Biosensor Characterisation for On-line Monitoring of Heavy Metals Pollutions in Waste Water Treatment Plant Effluents

International audienc

Raman Spectroscopy: Toward a Portable Food Quality-Warning System

Food waste is one of the main problems contributing to climate change as its piling up in landfills produces the greenhouse gas methane. Food waste occurs at every stage of food production; however, the major source of food waste occurs at businesses that supply food to consumers. Industry 4.0 technologies have shown promises in helping reduce food waste in food supply chains. However, more innovative technologies such as Raman spectroscopy holds great promise in helping reduce food waste, but this has largely been ignored in the literature. In this context, we propose a portable Raman platform to monitor food quality during transportation. The developed system was tested in conditions mimicking those present in a refrigerated truck by analyzing chicken samples stored at temperatures of 4 °C. Raman spectra were acquired for non-packaged and packaged samples over the duration of 30 days resulting in 6000 spectra. The analysis of Raman spectra revealed that the system was able to detect noticeable changes in chicken quality starting day six. The main Raman bands contributing to this change were amide I and tyrosine. The proposed system will offer the potential to reduce food losses during transportation by consistently checking the food quality over time

Développement de bioessais à l'aide de bactéries génétiquement modifiées pour la détection de polluants environnementaux

La Décision Européenne n2455/2001/CE a publié en 2001 une liste de 33 substances à détecter dans l eau. Nous avons abordé, avec des méthodes biologiques innovantes, le problème de la détection de deux de ces substances, le tributylétain (TBT), un biocide couramment utilisé dans les peintures antisalissures et interdit dans de nombreux pays, et l atrazine, un herbicide. La bactérie Escherichia coli TBT3, qui a été obtenue par mutagenèse aléatoire, est sensible aux organoétains mais son mécanisme d induction est inconnu. L étude au niveau moléculaire de cette bactérie a permis d aboutir à un mécanisme vraisemblable de la réponse au TBT, grâce à la localisation d un promoteur et d un nouveau régulateur transcriptionnel, responsables de l induction de la bioluminescence. Un dispositif innovant a été fabriqué pour distinguer les peintures interdites contenant du TBT de celle n en contenant pas. Un bioessai simple est proposé comme alternative aux analyses chimiques. En parallèle, une nouvelle bactérie, dont la bioluminescence est inductible par l atrazine, a été développée en détournant le système de dégradation de l atrazine provenant de la bactérie environnementale Pseudomonas sp. ADP. Cette nouvelle bactérie bioluminescente permettra une détection simple et rapide de l atrazine dans l eau et le sol.The European decision n2455/2001/EC published in 2001 a list of 33 priority substances to be detected in water. We addressed the problem of the detection with innovative biological methods for two substances, the tributyltin (TBT) a biocide broadly used in antifouling paint and now banned for many countries and the herbicide atrazine. The bacterium E. coli TBT3, which was made by random mutagenesis, is sensitive to organotins but its induction mechanism remained unknown. A comprehensive molecular study of this bacterium led to a plausible mechanism of TBT response, thanks to the localization of a promoter and a new transcriptional regulator YgaV involved in the induction of bioluminescence. An innovative device was made to distinguish prohibited paints containing TBT from those without TBT. A simple alternative bioassay is now proposed to the conventional analyses. In a second step, a new bacterium, whose bioluminescence was activated by atrazine, was also developed by using the biodegradation system of atrazine from an environmental bacterium Pseudomonas sp. ADP. This new bioluminescent bacteria will allow a simple and rapid way to detect atrazine both in water and in soil.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Optimisation des écoulements et des transferts de matière dans unmini-système de détection en continu de polluants par des bactéries bioluminescentes

La gestion et la qualité de l eau représentent une problématique réelle en Europe. L analyse des polluants et notamment les métaux lourds se fait essentiellement par des méthodes physico-chimiques sensibles mais coûteuses. D autres méthodes alternatives d analyse tels que les biocapteurs ont été développées pour leur simplicité et leur faible coût. Les travaux présentés dans cette étude se déroulent dans le cadre du développement de biocapteurs pour la mesure de polluants dans l environnement. Le sujet de thèse intervient plus particulièrement sur le développement d un biocapteur bactérien Lumisens III. Le système de mesure de ce dernier est composé d une carte contenant différentes bactéries immobilisées afin d assurer la mesure en ligne et simultanée de plusieurs polluants des eaux de l environnement. Cette carte de mesure a été présentée comme étant une boite noire délivrant un signal de bioluminescence dépendant de plusieurs paramètres environnementaux et microbiens rendant le processus très complexe et encore actuellement faiblement expliqué. La thèse a pour objectif d aboutir à une meilleure compréhension du phénomène de bioluminescence au sein même du biocapteur. Le travail s est déroulé en quatre parties: -L optimisation de la géométrie de la carte multipuits actuelle afin d augmenter les performances du biocapteur. - L étude des transferts au sein de la membrane d agarose contenant les bactéries immobilisées(modélisation de la diffusion du substrat, inducteur, oxygène). - L étude de la croissance bactérienne dans la matrice d immobilisationainsi que sa modélisation. - Une tentative de modélisation de la bioluminescence en milieu hétérogène. L originalité de cette étude réside dans le couplage des méthodes numériques et les phénomènes biologiques. Ces méthodes numériques nous ont aidé à mieux comprendre le processus de détection du biocapteur. De plus, à l heure actuelle et à notre connaissance, aucune étude n a été réalisée en système bactérien ouvert avec un flux de matière. Cette étude pourrait également trouver une extension dans différents types de biocapteurs enzymatiques ou immunologiques.TThe management and the quality of the water represent a real problem in Europe. The analysis of pollutants in particular heavy metals is essentially made by sensitive but expensive physico-chemical methods. Other alternative methods of analysis such as the biosensors were developed for their simplicity and their moderate cost. The studies presented in this study take place within the framework of the development of biosensors for the measurement of pollutants in the environment. This thesis is particularly devoted on the development of a bacterial biosensor Lumisens III. The measurement system of this last one consists of a card containing various immobilized bioluminescent bacteria to allow the on-line and simultaneous measurement of several water s pollutants. This card was presented as being a black box delivering a signal of bioluminescence dependent on several environmental and microbial parameters, the process of which is very complex and even at present weakly explained. The thesis aims to a better understanding of the phenomenon of bioluminescence inside the biosensor. The studies were divided in foour parts : - The optimization of the geometry of the current multiwell card to increase the performances of the biosensor. -The study of transfers within the agarose membrane containing immobilized bacteria (modelisation of the diffusion of the substrate, the pollutant and oxygen). - The study of the bacterial growth in the immobilisation matrix as well as its modelling. - An attempt of modelisation of the bioluminescence in heterogeneous medium. The originality of this study consist on the coupling of the numerical methods and the biological phenomena. These numerical methods helped us to understand better the detection process of the biosensor. Furthermore, at the moment and in our best knowledge, no study was realized in an open bacterial system with a flux of substances. This study could also find an extension in various types of enzymatic biosensors or immunological biosensors.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Systèmes biologiques pour le monitorage des eaux (aspects d'ingénieur, du laboratoire à la mesure sur le terrain)

NANTES-BU Sciences (441092104) / SudocSudocFranceF

The ygaVP Genes of Escherichia coli Form a Tributyltin-Inducible Operon▿ †

A tributyltin (TBT) luxAB transcriptional fusion in Escherichia coli revealed that a TBT-activated promoter is located upstream of two cotranscribed orphan genes, ygaV and ygaP. We demonstrate that transcription from the promoter upstream of ygaVP is constitutive in a ygaVP mutant, suggesting that YgaV is an autoregulated, TBT-inducible repressor

Development and Automation of a Bacterial Biosensor to the Targeting of the Pollutants Toxic Effects by Portable Raman Spectrometer

Water quality monitoring requires a rapid and sensitive method that can detect multiple hazardous pollutants at trace levels. This study aims to develop a new generation of biosensors using a low-cost fiber-optic Raman device. An automatic measurement system was thus conceived, built and successfully tested with toxic substances of three different types: antibiotics, heavy metals and herbicides. Raman spectroscopy provides a multiparametric view of metabolic responses of biological organisms to these toxic agents through their spectral fingerprints. Spectral analysis identified the most susceptible macromolecules in an E. coli model strain, providing a way to determine specific toxic effects in microorganisms. The automation of Raman analysis reduces the number of spectra required per sample and the measurement time: for four samples, time was cut from 3 h to 35 min by using a multi-well sample holder without intervention from an operator. The correct classifications were, respectively, 99%, 82% and 93% for the different concentrations of norfloxacin, while the results were 85%, 93% and 81% for copper and 92%, 90% and 96% for 3,5-dichlorophenol at the three tested concentrations. The work initiated here advances the technology needed to use Raman spectroscopy coupled with bioassays so that together, they can advance field toxicological testing