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Isolation of oxalotrophic bacteria able to disperse on fungal mycelium

2013, Bravo, Daniel, Cailleau, Guillaume, Bindschedler, Saskia, Simon, Anaele, Job, Daniel, Verrecchia, Eric, Junier, Pilar

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Use of an isothermal microcalorimetry assay to characterize microbial oxalotrophic activity

2011, Bravo, Daniel, Braissant, Olivier, Solokhina, Anna, Clerc, Martin, Daniels, Alma U., Verrecchia, Eric, Junier, Pilar

Isothermal microcalorimetry (IMC) has been used in the past to monitor metabolic activities in living systems. A few studies have used it on ecological research. In this study, IMC was used to monitor oxalotrophic activity, a widespread bacterial metabolism found in the environment, and particularly in soils. Six model strains were inoculated in solid angle media with K-oxalate as the sole carbon source. Cupriavidus oxalaticus, Cupriavidus necator, and Streptomyces violaceoruber presented the highest activity (91, 40, and 55 μW, respectively) and a maximum growth rate (μmax h−1) of 0.264, 0.185, and 0.199, respectively, among the strains tested. These three strains were selected to test the incidence of different oxalate sources (Ca, Cu, and Fe-oxalate salts) in the metabolic activity. The highest activity was obtained in Ca-oxalate for C. oxalaticus. Similar experiments were carried out with a model soil to test whether this approach can be used to measure oxalotrophic activity in field samples. Although measuring oxalotrophic activity in a soil was challenging, there was a clear effect of the amendment with oxalate on the metabolic activity measured in soil. The correlation between heat flow and growth suggests that IMC analysis is a powerful method to monitor bacterial oxalotrophic activity.

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Metaproteomics and ultrastructure characterization of Komagataeibacter spp. involved in high-acid spirit vinegar production

, Andrés-Barrao, Cristina, Saad, Maged M, Cabello Ferrete, Elena, Bravo, Daniel, Chappuis, Marie-Luise, Ortega Pérez, Ruben, Junier, Pilar, Perret, Xavier, Barja, François

Acetic acid bacteria (AAB) are widespread microorganisms in nature, extensively used in food industry to transform alcohols and sugar alcohols into their corresponding organic acids. Specialized strains are used in the production of vinegar through the oxidative transformation of ethanol into acetic acid. The main AAB involved in the production of high-acid vinegars using the submerged fermentation method belong to the genus Komagataeibacter, characterized by their higher ADH stability and activity, and higher acetic acid resistance (15-20%), compared to other AAB.
In this work, the bacteria involved in the production of high-acid spirit vinegar through a spontaneous acetic acid fermentation process was studied. The analysis using a culture-independent approach revealed a homogeneous bacterial population involved in the process, identified as Komagataeibacter spp. Differentially expressed proteins during acetic acid fermentation were investigated by using 2DDIGE and mass spectrometry. Most of these proteins were functionally related to stress response, the TCA cycle and different metabolic processes. In addition, scanning and transmission electron microscopy and specific staining of polysaccharide SDS-PAGE gels confirmed that Komagataeibacter spp. lacked the characteristic polysaccharide layer surrounding the outer membrane that has been previously reported to have an important role in acetic acid resistance in the genus Acetobacter.

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Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments

2013, Bravo, Daniel, Martin, Gaëtan, David, Maude M, Cailleau, Guillaume, Verrecchia, Eric, Junier, Pilar

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Isolation and characterization of oxalotrophic bacteria from tropical soils

, Bravo, Daniel, Braissant, Olivier, Cailleau, Guillaume, Verrecchia, Eric, Junier, Pilar

The oxalate–carbonate pathway (OCP) is a biogeochemical set of reactions that involves the conversion of atmospheric CO2 fixed by plants into biomass and, after the biological recycling of calcium oxalate by fungi and bacteria, into calcium carbonate in terrestrial environments. Oxalotrophic bacteria are a key element of this process because of their ability to oxidize calcium oxalate. However, the diversity and alternative carbon sources of oxalotrophs participating to this pathway are unknown. Therefore, the aim of this study was to characterize oxalotrophic bacteria in tropical OCP systems from Bolivia, India, and Cameroon. Ninety-five oxalotrophic strains were isolated and identified by sequencing of the 16S rRNA gene. Four genera corresponded to newly reported oxalotrophs (Afipia, Polaromonas, Humihabitans, and Psychrobacillus). Ten strains were selected to perform a more detailed characterization. Kinetic curves and microcalorimetry analyses showed that Variovorax soli C18 has the highest oxalate consumption rate with 0.240 μM h-1. Moreover, Streptomyces achromogenes A9 displays the highest metabolic plasticity. This study highlights the phylogenetic and physiological diversity of oxalotrophic bacteria in tropical soils under the influence of the oxalate–carbonate pathway.

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Assessing the diversity and metabolism of oxalotrophic bacteria in tropical soils

2013, Bravo, Daniel, Junier, Pilar

Climate change is increasing as a consequence of elevated concentrations of CO2 in the atmosphere. Among the scientific strategies proposed to tackle the effect of elevated CO2, the biogeochemical oxalate-carbonate pathway (OCP) occurring in terrestrial habitats, appears to be important since it is considered as a potential carbon sink. This process occurs naturally in Earth, with a particular importance in tropical forests. Previous studies were carried out in a subtropical forest to evaluate the precipitation and biomineralization of carbonate due to biologic (microbial oxalotrophy) activity. However, the knowledge about biodiversity and the metabolic rates of oxalate consumption, as well as the effect of microbial interactions over the OCP at several tropical soils was poorly described. Therefore, the aim of this thesis was to assess the diversity and metabolism of oxalotrophic bacteria related with the OCP in tropical habitats. This document represents the first detailed study about metabolism and diversity of oxalotrophic bacteria found in three tropical soils in Bolivia, Indian, and Cameroon, implicated in oxalate-carbonate transformations. At those sampling sites, oxalate producing trees (oxalogenic trees) were assessed by biotic and abiotic treats influencing the pathway occurring there. The manuscript is organized in seven chapters. The first two chapters deal with the development and application of analytical and molecular techniques, such as isothermal microcalorimetry (IMC) and BrdU labeling DNA - DGGE, to study metabolism and diversity of model and active environmental oxalotrophic bacteria. The relevance of active oxalotrophic bacteria is highlighted. For instance, the ecological role of relatives to Kribbella phylotypes and related actinobacteria within the oxalotrophic group found in Cameroon is discussed at the end of chapter three. Moreover, the following chapter deal with a collection of oxalotrophic bacteria isolated from soil samples recovered in field trips performed at Bolivia, India, and Cameroon, where oxalogenic trees were found. The chapter includes a complete characterization of ten oxalotrophs, with the interest to understand their capability to consume oxalate as sole carbon and energy source, as well as, their metabolic plasticity by the consumption of other carbon substrates. High oxalate consumption rates were observed for strains such as Variovorax soil C18, Lysobacter sp. A8, Agrobacterium sp. B23, and Streptomyces achromogenes A9. Chapter five describe the development of a new technique of isolation of autochthonous couples of bacteria and fungi from soil implied in oxalotrophy. The case of the fungus Trichoderma sp. and eight oxalotrophic bacteria obtained from soils samples influenced by oxalogenic trees in Cameroon is discussed. Furthermore, a global discussion including a comparison of abundance and vertical distribution of oxalotrophic bacteria through soil profiles influenced by oxalate-producer trees is part of the chapter six. Biotic and abiotic treats are correlated statistically to compare and understand the OCP systems occurring in India and Cameroon. The contribution to the knowledge in oxalotrophic diversity and metabolism relative to the oxalate-carbonate pathway occurring in tropical soils, as well as, the perspectives of this work in CO2 management are presented at the end of chapter six. In chapter seven is exposed a complete revision of the interactions between soil pH, fungi and bacteria as key actors at the geo-biological interface of the pathway, put it all in evidences through microcosms experiments.

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Fungi, bacteria and soil pH: the oxalate–carbonate pathway as a model for metabolic interaction

, Martin, Gaëtan, Guggiari, Matteo, Bravo, Daniel, Zopfi, Jakob, Cailleau, Guillaume, Aragno, Michel, Job, Daniel, Verrecchia, Eric, Junier, Pilar

The oxalate–carbonate pathway involves the oxidation of calcium oxalate to low-magnesium calcite and represents a potential long-term terrestrial sink for atmospheric CO2. In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non-sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.