Benzo(a)pyrene inhibits the role of the bioturbator Tubifex tubifex in river sediment biogeochemistry

International audienceThe interactions between invertebrates and micro-organisms living in streambed sediments often play key roles in the regulation of nutrient and organic matter fluxes in aquatic ecosystems. However, benthic sedi- ments also constitute a privileged compartment for the accumulation of persistent organic pollutants such as PAHs or PCBs that may affect the diversity, abundance and activity of benthic organisms. The objective of this study was to quantify the impact of sediment contamination with the PAH benzo(a)pyrene on the in- teraction between micro-organisms and the tubificid worm, Tubifex tubifex, which has been recognized as a major bioturbator in freshwater sediments. Sedimentary microcosms (slow filtration columns) contaminated or not with benzo(a)pyrene (3 tested concentrations: 0, 1 and 5 mg kg−1) at the sediment surface were in- cubated under laboratory conditions in the presence (100 individuals) or absence of T. tubifex. Although the surface sediment contaminations with 1 mg kg−1 and 5 mg kg−1 of benzo(a)pyrene did not affect tubificid worm survival, these contaminations significantly influenced the role played by T. tubifex in biogeochemical processes. Indeed, tubificid worms stimulated aerobic respiration, denitrification, dehydrogenase and hydrolytic activities of micro-organisms in uncontaminated sediments whereas such effects were inhibited in sediments polluted with benzo(a)pyrene. This inhibition was due to contaminant-induced changes in bioturbation (and especially bio-irrigation) activities of worms and their resulting effects on microbial processes. This study reveals the importance of sublethal concentrations of a contaminant on ecological processes in river sediments through affecting bioturbator-microbe interactions. Since they affect microbial processes involved in water purification processes, such impacts of sublethal concentrations of pollutants should be more often considered in ecosystem health assessment

How metabolomics is used to support the MetaPath solution through metabolic profiling of cheese fermentation industry ecosystem models ?

International audienceConsumer expectations and societal evolutions are at the heart of the concerns of fermentation professionals. The organoleptic properties of their products are closely correlated to the manufacturing process but also to its composition specifically the microbial ecosystems introduced to produce this fermentation. In this context, the Bpifrance MetaPath project aims to reconstruct the metabolic maps of these microbial ecosystems in order to guide the industrialist in the selection of microbial strains at the time of product design. As a partner, the MetaToul platform aims to perform metabolic profiling of ecosystems in the cheese model of industrial partner Bel. The sample preparation and analysis methods developed on the MetaToul platform on this complex matrix allow a complete and precise study of the central and energetic metabolism (~80 metabolites) of these ecosystem models. It was validated with the addition of IDMS on repeatability parameters, extraction yield and matrix effect. The samples produced for metabolomics are analyzed on the following analytical systems: ion chromatography coupled to an LTQ-Orbitrap-Velos (Thermo) for the analysis of central energetic metabolism, liquid chromatography coupled to a QExactive+ (Thermo) for the analysis of amino acids, coenzymes A and the mevalonate pathway. The metabolic profiling data produced on the different ecosystem models of the partner will be integrated with others omics data into the MetaPath solution by Abolis/ Microbiome Studio partner in order to reconstruct active metabolic maps, true identity cards of the metabolic activity of microbial ecosystems in the fermentation of cheese matrix

An optimization method for untargeted MS-based isotopic tracing investigations of metabolism

International audienceIntroduction Stable isotope tracer studies are increasingly applied to explore metabolism from the detailed analysis of tracer incorporation into metabolites. Untargeted LC/MS approaches have recently emerged and provide potent methods for expanding the dimension and complexity of the metabolic networks that can be investigated. A number of software tools have been developed to process the highly complex MS data collected in such studies; however, a method to optimize the extraction of valuable isotopic data is lacking. Objectives To develop and validate a method to optimize automated data processing for untargeted MS-based isotopic tracing investigations of metabolism. Methods The method is based on the application of a suitable reference material to rationally perform parameter optimization throughout the complete data processing workflow. It was applied in the context of 13 C-labelling experiments and with two different software, namely geoRge and X13CMS. It was illustrated with the study of a E. coli mutant impaired for central metabolism. Results The optimization methodology provided significant gain in the number and quality of extracted isotopic data, independently of the software considered. Pascal triangle samples are well suited for such purpose since they allow both the identification of analytical issues and optimization of data processing at the same time. Conclusion The proposed method maximizes the biological value of untargeted MS-based isotopic tracing investigations by revealing the full metabolic information that is encoded in the labelling patterns of metabolites

Metabolic profiles of complex fermentation matrices from cream and bakery industry

International audienceIntroduction Microbial ecosystems are key factors in food production by fermentation. The metabolic potential of ecosystems is very important and varies depending on the ecosystem composition and the metabolic capacity of the different partners. The construction of the metabolic map of an ecosystem is an important tool to understand, develop and use these metabolic capacities. In this context the MetaPath Bpi France project aims to develop an integrated solution allowing this modeling. In this context, the MetaToul platform aims to develop methods adapted to different food matrices for study of the metabolism, the most accurate and global possible of these different cellular ecosystems. Our main action is first of all to develop, on these complex fermented cream and sourdoughs, the extraction and global analysis of the central and energetic metabolism

How metabolomics is used to support the MetaPath solution through metabolic profiling of sourdough fermentation industry ecosystem models?

Consumer expectations and societal evolutions are at the heart of the concerns of fermentation professionals. The organoleptic properties of their products are closely correlated to its manufacturing process but also to its composition specifically the microbial ecosystems introduced to produce this fermentation. In this context, the Bpifrance MetaPath project aims to reconstruct the metabolic maps of these microbial ecosystems in order to guide the industrialist in the selection of microbial strains at the time of product design. As a partner, the MetaToul platform aims to perform metabolic profiling of ecosystems in the sourdough model of industrial partner Lesaffre. The sample preparation and analysis methods developed on the MetaToul platform on this complex matrix allow a complete and precise study of the central and energetic metabolism (~80 metabolites) of these ecosystem models. It was validated with the addition of IDMS on repeatability parameters, extraction yield and matrix effect. The samples produced for metabolomics are analyzed on the following analytical systems: ion chromatography coupled to an LTQ-Orbitrap-Velos (Thermo) for the analysis of central energetic metabolism, liquid chromatography coupled to a QExactive+ (Thermo) for the analysis of amino acids, coenzymes A and the mevalonate pathway. The metabolic profiling data produced on the different ecosystem models of the partner will be integrated with others omics data into the MetaPath solution by Abolis/ Microbiome Studio partner in order to reconstruct active metabolic maps, true identity cards of the metabolic activity of microbial ecosystems in the fermentation of sourdough matrix

Miniaturization of a metabolomics and fluxomics sample preparation workflow

International audienceOne of the current challenges in metabolomics and fluxomics is to gain access to the cellular heterogeneity to have access to subpopulations of cells or even of a single cell metabolism. In its second component, the national metabolomics and fluxomics infrastructure MetaboHUB is developing a work package with the objective of being able to perform single cell experiments in metabolomics and fluxomics. Unlike other omics like transcriptomics, Single-cell metabolomics is still in its infancy and only very few attempts of single cell fluxomics have been reported so far. In this poster, focus will be done on the developments realized for metabolomics and fluxomic sample preparation step. Indeed, methods conventionally used involve working with several hundred microliters or even several milliliters. An intermediate step of this workpackage is to be able to work on small volumes of samples in order to analyze matrices for which we have few amounts available or to concentrate classical samples and have access to minority metabolites. For this purpose, we adapted our current robotic systems in order to allow precision work with smaller volumes. The developments made allow us today to aspirate and dispense 2 µL with a CV less than 10%. In a second hand, developments of new robotic system protocols were done in order to prepare miniaturized NMR samples. With the use of new capillary needles, we can automatically filled NMR tubes of 1.7 and 1mm inner diameters. This opens up new perspectives, notably by reducing the volume of samples required for NMR analysis

Sink/Source Balance of Leaves Influences Amino Acid Pools and Their Associated Metabolic Fluxes in Winter Oilseed Rape (Brassica napus L.)

International audienceNitrogen remobilization processes from source to sink tissues in plants are determinant for seed yield and their implementation results in a complete reorganization of the primary metabolism during sink/source transition. Here, we decided to characterize the impact of the sink/source balance on amino acid metabolism in the leaves of winter oilseed rape grown at the vegetative stage. We combined a quantitative metabolomics approach with an instationary N-15-labeling experiment by using [N-15](L)-glycine as a metabolic probe on leaf ranks with a gradual increase in their source status. We showed that the acquisition of the source status by leaves was specifically accompanied by a decrease in asparagine, glutamine, proline and S-methyl-l-cysteine sulphoxide contents and an increase in valine and threonine contents. Dynamic analysis of N-15 enrichment and concentration of amino acids revealed gradual changes in the dynamics of amino acid metabolism with respect to the sink/source status of leaf ranks. Notably, nitrogen assimilation into valine, threonine and proline were all decreased in source leaves compared to sink leaves. Overall, our results suggested a reduction in de novo amino acid biosynthesis during sink/source transition at the vegetative stage

Velamo do Campo : Its Volatile Constituents, Secretory Elements, and Biological Activity

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IsoCor: isotope correction for high-resolution MS labeling experiments

International audienceMass spectrometry (MS) is widely used for isotopic studies of metabolism and other (bio)chemical processes. Quantitative applications in systems and synthetic biology require to correct the raw MS data for the contribution of naturally occurring isotopes. Several tools are available to correct low-resolution MS data, and recent developments made substantial improvements by introducing resolution-dependent correction methods, hence opening the way to the correction of high-resolution MS (HRMS) data. Nevertheless, current HRMS correction methods partly fail to determine which isotopic species are resolved from the tracer isotopologues and should thus be corrected. We present an updated version of our isotope correction software (IsoCor) with a novel correction algorithm which ensures to accurately exploit any chemical species with any isotopic tracer, at any MS resolution. IsoCor v2 also includes a novel graphical user interface for intuitive use by end-users and a command-line interface to streamline integration into existing pipelines

Development of high throughput miniaturized robotic platforms for metabolism investigations of microorganisms

During the past few years, we have developed a fully integrated solution to analyze the fluxome (i.e. in vivo reaction rate associated with a cellular network of an organism), which combines two robotic cultivation and sampling workstation for 13C-labelling experiments with MS and NMR-based isotopic profiling, and tools for processing and interpreting isotopic data.First of all, we developed in collaboration with TECAN, a platform for automated HT-fluxome profiling of metabolic variants. Controlled by two software, this robotic system prepares, runs, monitors and controls the fermentations, and adjusts the pH, temperature and stirrer speed for 48 micro-scale (10 ml) fermentations in parallel. The robotic system also features fully automated metabolite harvesting and extraction (e.g. cell pellets, supernatants and intracellular metabolites) for downstream analysis.Drastically increasing the quantity of biological samples produced, a second robotic platform has been acquired to fully automatize the preparation of biological samples for analysis by liquid chromatography coupled to mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The device is able to handle a complete range of sample racks as well as carrying out various types of manipulation such as dilution, aliquoting, addition of buffer and internal standard, filtration, centrifugation, sample pre-treatment (i.e SPE) all while ensuring traceability of all samples.Recently, we focus our development on the handling of low-volume or small-quantity samples in order to analyze matrices for which we have few amounts available or to concentrate classical samples and have access to minority metabolites. We have adapted our robotic platform to pipette a few microliters (2µL) of sample while maintaining excellent precision (CV<10%), and equipped the pipetting arm with capillary needles to fill 1.7mm internal diameter NMR tubes. This opens up new prospects, including the possibility of studying the metabolism of new sample niches such as spheroids or others extracts with very low volumes.We have now a complete high-throughput, miniaturized workflow for microorganisms cultivations, preparing and analyzing miniaturized samples, as well as bioinformatics tools for processing the large datasets generated. This entire workflow is now available to scientists wishing to carry out targeted or non-targeted metabolomics and fluxomic experiments