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Background Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete Trichoderma reesei, but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the T. reesei enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity. Results In this study, the enzymatic cocktails secreted by five strains from the genus Aspergillus (Aspergillus japonicus strains BRFM 405, 1487, 1489, 1490 and Aspergillus niger strain BRFM 430) were tested for their ability to boost a T. reesei reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from Aspergillus aculeatus (AaAA16) was produced to high level in Pichia pastoris. LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of T. reesei CBHI on cellulosic substrates. Conclusions Although Aspergillus spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries. Electronic supplementary material The online version of this article (10.1186/s13068-019-1394-y) contains supplementary material, which is available to authorized users.

Enzymatic degradation of plant biomass is of growing interest for the development of a sustainable bio-based industry. Filamentous fungi, which degrade complex and recalcitrant plant polymers, are proficient secretors of enzymes acting on the lignocellulose composite of plant cell walls in addition to starch, the main carbon storage reservoir. In this review, we focus on the identification of lytic polysaccharide monooxygenases (LPMOs) and their redox partners in fungal secretomes to highlight the biological functions of these remarkable enzyme systems and we discuss future trends related to LPMO-potentiated bioconversion.

International audience; Alternaria brassicicola is a necrotrophic fungus causing black spot disease andis an economically important seed-borne pathogen of cultivated brassicas. Seedtransmission is a crucial component of its parasitic cycle as it promotes long-termsurvival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A.brassicicola pathosystem, showed that the level of susceptibility of the fungus to waterstress strongly influenced its seed transmission ability. In this study, we gained furtherinsights into the mechanisms involved in the seed infection process by analyzing thetranscriptomic and metabolomic responses of germinated spores of A. brassicicolaexposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteinsthat are assumed to be involved in cellular dehydration tolerance, was established in A.brassicicola based on the expression data and additional structural and biochemicalcriteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-likeproteins showed that they were affected in their transmission to A. thaliana seeds,although their aggressiveness on host vegetative tissues remained intact.

L’UCO de Guingamp vous propose de nous réunir lors de la 3ème édition de notre évènement U’COOK-INNOVATION autour de la thématique les « Les Produits Fermentés » : UN THÈME FÉDÉRATEUR ET TENDANCE Il nous renvoie à notre RESPONSABILITE de développer une consommation saine, équilibrée & durable dans le monde UN AXE STRATÉGIQUE DE DÉVELOPPEMENT pour de nombreuses entreprises face à la croissance de la demande des consommateurs PLUSIEURS QUESTIONS se posent alors sur ces produits : la qualité, le goût, le prix, l’aspect nutrition/santé, la certification, leurs limites… 6 INTERVENANTS EXPERTS DANS LEUR MÉTIER expliqueront le rôle et les enjeux de la fermentation : marché, coûts, limites, implications…Un focus sera fait sur les différentes fermentations (procédés, points communs et différences) sur le microbiote digestif et sur les bienfaits immunitaires et digestifs, sans oublier les limites d’une consommation excessive.

Filamentous fungi are the most common industrial xylanase producers. In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation affected the molecular mass of the recombinant xylanase, resulting in an apparent molecular weight larger than 60 kDa, whereas after deglycosylation, the recombinant XynA displayed a molecular mass of 20 kDa. The deglycosylated xylanase was purified by ion exchange chromatography and reached 185-fold of purification. The enzyme was optimally active at 55 degrees C and pH 5 and stable over a broad pH range (3-9). It retained more than 80% of the original activity after 24 h. It conserved around 80% of the original activity after pre-incubation at 40 degrees C for 6 h. With birchwood xylan as substrate, the enzyme showed a K-m of 5.2 mg/mL, and K-cat of 245 per s. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the re-XynA could be useful for variety of biotechnological applications.

[Background] Lavender (Lavandula angustifolia) and lavandin (a sterile hybrid of L. angustifolia × L. latifolia) essential oils are among those most commonly used in the world for various industrial purposes, including perfumes, pharmaceuticals and cosmetics. The solid residues from aromatic plant distillation such as lavender- and lavandin-distilled straws are generally considered as wastes, and consequently either left in the fields or burnt. However, lavender- and lavandin-distilled straws are a potentially renewable plant biomass as they are cheap, non-food materials that can be used as raw feedstocks for green chemistry industry. The objective of this work was to assess different pathways of valorization of these straws as bio-based platform chemicals and fungal enzymes of interest in biorefinery., [Results] Sugar and lignin composition analyses and saccharification potential of the straw fractions revealed that these industrial by-products could be suitable for second-generation bioethanol prospective. The solvent extraction processes, developed specifically for these straws, released terpene derivatives (e.g. τ-cadinol, β-caryophyllene), lactones (e.g. coumarin, herniarin) and phenolic compounds of industrial interest, including rosmarinic acid which contributed to the high antioxidant activity of the straw extracts. Lavender and lavandin straws were also suitable inducers for the secretion of a wide panel of lignocellulose-acting enzymes (cellulases, hemicellulases and oxido-reductases) from the white-rot model fungus Pycnoporus cinnabarinus. Interestingly, high amounts of laccase and several lytic polysaccharide monooxygenases were identified in the lavender and lavandin straw secretomes using proteomics., [Conclusions] The present study demonstrated that the distilled straws of lavender and lavandin are lignocellulosic-rich materials that can be used as raw feedstocks for producing high-added value compounds (antioxidants, aroma) and fungal oxidative enzymes, which represent opportunities to improve the decomposition of recalcitrant lignocellulose into biofuel. Hence, the structure and the physico-chemical properties of these straws clearly open new perspectives for use in biotechnological processes involving especially filamentous fungi. These approaches represent sustainable strategies to foster the development of a local circular bioeconomy., This work was funded by the French National Institute for Agricultural and Sea Products (FranceAgriMer), the company Naturamole (Susville, France), and the French Pole of Competitiveness for Perfumes, Flavors and Fragrances (PASS pole, Grasse, France).

The yeast trehalose-6-phosphate synthase (Tps1) catalyzes the formation of trehalose-6-phosphate (T6P) in trehalose synthesis. Besides, Tps1 plays a key role in carbon and energy homeostasis in this microbial cell, as shown by the well documented loss of ATP and hyper accumulation of sugar phosphates in response to glucose addition in a mutant defective in this protein. The inability of a Saccharomyces cerevisiae tps1 mutant to cope with fermentable sugars is still a matter of debate. We reexamined this question through a quantitative analysis of the capability of TPS1 homologues from different origins to complement phenotypic defects of this mutant. Our results allowed to classify this complementation in three groups. A first group enclosed TPS1 of Klyveromyces lactis with that of S. cerevisiae as their expression in Sctps1 cells fully recovered wild type metabolic patterns and fermentation capacity in response to glucose. At the opposite was the group with TPS1 homologues from the bacteria Escherichia coli and Ralstonia solanacearum, the plant Arabidopsis thaliana and the insect Drosophila melanogaster whose metabolic profiles were comparable to those of a tps1 mutant, notably with almost no accumulation of T6P, strong impairment of ATP recovery and potent reduction of fermentation capacity, albeit these homologous genes were able to rescue growth of Sctps1 on glucose. In between was a group consisting of TPS1 homologues from other yeast species and filamentous fungi characterized by 5 to 10 times lower accumulation of T6P, a weaker recovery of ATP and a 3-times lower fermentation capacity than wild type. Finally, we found that glucose repression of gluconeogenic genes was strongly dependent on T6P. Altogether, our results suggest that the TPS protein is indispensable for growth on fermentable sugars, and points to a critical role of T6P as a sensing molecule that promotes sugar fermentation and glucose repression..

AGAP : équipe GE2pop; Genotype by genotype indirect genetic effects (G x G IGEs) occur when the phenotype of an individual is influenced by an interaction between its own genotype and those of neighbour individuals. Little is known regarding the relative importance of G x G IGEs compared with other forms of direct and indirect genetic effects. We quantified the relative importance of IGEs in the filamentous fungus Aspergillus nidulans, a species in which IGEs are likely to be important as air-borne social interactions are known to affect growth. We used a collection of distantly related wild isolates, lab strains and a set of closely related mutation accumulation lines to estimate the contribution of direct and indirect genetic effects on mycelium growth rate, a key fitness component. We found that indirect genetic effects were dominated by G x G IGEs that occurred primarily between a focal genotype and its immediate neighbour within a vertical stack, and these accounted for 11% of phenotypic variation. These results indicate that G x G IGEs may be substantial, at least in some systems, and that the evolutionary importance of these interactions may be underappreciated, especially in microbes. We advocate for a wider use of the IGE framework in both applied (for example, choice of varietal mixtures in plant breeding) and evolutionary genetics (kin selection/kin competition studies).

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells. While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

Cadophora malorum Mo12 was isolated from the Rainbow hydrothermal site on the Mid-Atlantic Ridge. We present the draft genome sequence of this filamentous fungal strain, which has high biotechnological potentials as revealed by the presence of genes encoding biotechnologically important enzymes and genes involved in the synthesis of secondary metabolites.

International audience; Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharornyces cerevisiae is used namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast its low growth rate and low ethanol productivity hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the gamma-aminobutyric acid shunt, and an excess in NADPH regeneration.

Biotechnological Innovations through Fungi

The relationship between the physiological state of fungi and the response of their functional system to metals is not known, limiting the use of fungal enzymes as tools for assessing metal ecotoxicity in terrestrial ecosystems. The present study attempts to establish how the development phases modulate the secretion of enzymes in the filamentous fungus Trametes versicolor after exposure to Cu. For that purpose, extracellular hydrolases (acid and alkaline phosphatases, aryl-sulfatase, beta-glucosidase, beta-galactosidase, and N-acetyl-beta-glucosaminidase) and oxidoreductases (laccase, manganese and lignin peroxidases) were monitored in liquid cultures for 2 weeks. Copper was added during either the growth or the stationary phases at 20 or 200 ppm. Results of the present study showed that Cu at the highest concentration modifies the secretion of enzymes, regardless of the development phase to which the fungus was exposed. However, the sensitivity of enzyme responses to Cu depended on the phase development and the type of secreted enzyme. In a general way, the production of hydrolases was decreased by Cu, whereas that of oxidoreductases was highly increased. Furthermore, lignin peroxidase was not detected in control cultures and was specifically produced in the presence of Cu. In conclusion, fungal oxidoreductases may be enzymatic biomarkers of copper exposure for ecotoxicity assessment.

Background The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood. Results In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). Conclusions This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0274-3) contains supplementary material, which is available to authorized users.

International audience; Marine ecosystems in active volcanic areas are regularly subject to natural hazardssuch as being covered by incandescent lava flows, acoustic phenomena caused bymicroseisms, temporary changes in physicochemical conditions of water bodiesand exceptional elevations of temperature. The Piton de la Fournaise (ReunionIsland) is one of the most active effusive volcanoes in the world with 27eruptions between 1998 and 2007 and a mean frequency, over a century, of aneruptive phase every 9 months. It provides a natural laboratory to study thecolonization of a blank substrate. Therefore, a project called BIOLAVE waslaunched in 2010 in order to study species and habitat diversity on the coastalmarine area of the volcano, including the first taxonomic inventory for thesoutheast side of the Island [1].One hundred fungal isolates were obtained from 1977 lava flow (-25m depth),2004 lava flow (-70m) and free water (-70m) as preliminary sampling. Density ofpropagules was as follows: 0.085/g free water (-70m), 20/g 2004 lava (–70m) and80/g 1977 lava (-25m) (MEA, PDA and Sabouraud media). These isolates shouldnow be identified using phenotypic characteristics and molecular biology.

Background Enzymatic breakdown of lignocellulosic biomass is a known bottleneck for the production of high-value molecules and biofuels from renewable sources. Filamentous fungi are the predominant natural source of enzymes acting on lignocellulose. We describe the extraordinary cellulose-deconstructing capacity of the basidiomycete Laetisaria arvalis, a soil-inhabiting fungus. Results The L. arvalis strain displayed the capacity to grow on wheat straw as the sole carbon source and to fully digest cellulose filter paper. The cellulolytic activity exhibited in the secretomes of L. arvalis was up to 7.5 times higher than that of a reference Trichoderma reesei industrial strain, resulting in a significant improvement of the glucose release from steam-exploded wheat straw. Global transcriptome and secretome analyses revealed that L. arvalis produces a unique repertoire of carbohydrate-active enzymes in the fungal taxa, including a complete set of enzymes acting on cellulose. Temporal analyses of secretomes indicated that the unusual degradation efficiency of L. arvalis relies on its early response to the carbon source, and on the finely tuned sequential secretion of several lytic polysaccharide monooxygenases and hydrolytic enzymes targeting cellulose. Conclusions The present study illustrates the adaptation of a litter-rot fungus to the rapid breakdown of recalcitrant plant biomass. The cellulolytic capabilities of this basidiomycete fungus result from the rapid, selective and successive secretion of oxidative and hydrolytic enzymes. These enzymes expressed at critical times during biomass degradation may inspire the design of improved enzyme cocktails for the conversion of plant cell wall resources into fermentable sugars. Electronic supplementary material The online version of this article (doi:10.1186/s13068-014-0143-5) contains supplementary material, which is available to authorized users.

The objectives of this thesis are, on one hand, to develop an efficient system of agitation in terms of oxygen transfer and shear stress, suited for the fermentation of yeasts, bacteria and filamentous fungi, on the other hand, to develop a process of spore production in a liquid medium. This new process will be used instead of the solid culture in Roux flasks used by DSM at the beginning of the PhD program. In a first step, we have selected “Elephant Ears” impellers as stirrers; its effectiveness of oxygen transfer was characterized in a broad range of stirring speeds and airflow rates. The data obtained were modeled and a predictive tool for KLa and process extrapolation was developed. The extrapolation is based on the non-geometrical similarity and the coefficient of oxygen transfer (KLa) was kept constant between scales. The effectiveness of the new stirrer was validated in a pilot-scale in the presence of the cultures of bacteria, yeasts and filamentous fungi. Besides the studies on the stirrer, a process in a liquid medium of filamentous fungi was developed. Thus, the fermentation medium and the operating conditions of each steps of the fermentation (germination, vegetative growth, and sporulation) were optimized. This new process makes possible to obtain up to 200.106 cells per milliliter of culture on both laboratory and pilot scale (900L). Finally, the data gathered on the pilot scale was used to extrapolate this process to the industrial-scale of 10m3, Les objectifs de cette thèse sont, d’une part, de développer un système d’agitation performant en termes de transfert d‘oxygène et de forces de cisaillement afin qu’il soit adapté aux cultures de levures, de bactéries et de champignons filamenteux. D’autre part, de mettre au point un procédé de production des spores de champignons en milieu liquide pour remplacer la culture sur milieu solide (boîte de roux) utilisée au démarrage de la thèse par la société DSM. Ces travaux ont permis de sélectionner le mobile type « oreilles d’éléphant » comme système d’agitation. Son efficacité de transfert d’oxygène a été caractérisée dans une large gamme de débits d’air et de vitesses d’agitation. Les données obtenues ont été modélisées pour développer un outil de prédiction du KLa et d’extrapolation de procédé d’agitation. L’approche utilisée pour l’extrapolation est basée sur la similitude non géométrique et le maintien du coefficient de transfert d’oxygène (KLa) constant entre les échelles. L’efficacité du nouveau mobile d’agitation a été validée à l’échelle pilote en présence des cultures de bactéries, de levures et de champignons. Parallèlement au choix du mobile d’agitation, un procédé de production de spores de champignons filamenteux en milieu liquide a été développé. Ainsi, le milieu de culture et les conditions opératoires de chaque étape de la fermentation (germination, croissance végétative et sporulation) ont été optimisés. Ce nouveau procédé permet d’obtenir jusqu’à 200.106 cellules par millilitre de culture aussi bien à l’échelle du laboratoire que pilote (900L). Il a été aussi validé en présence de cultures de levures et de bactéries. Sur le même principe, les données obtenues à l’échelle pilote ont permis de développer un outil d’extrapolation à l’échelle industrielle de 10 m3

The objectives of this thesis are, on one hand, to develop an efficient system of agitation in terms of oxygen transfer and shear stress, suited for the fermentation of yeasts, bacteria and filamentous fungi, on the other hand, to develop a process of spore production in a liquid medium. This new process will be used instead of the solid culture in Roux flasks used by DSM at the beginning of the PhD program. In a first step, we have selected “Elephant Ears” impellers as stirrers; its effectiveness of oxygen transfer was characterized in a broad range of stirring speeds and airflow rates. The data obtained were modeled and a predictive tool for KLa and process extrapolation was developed. The extrapolation is based on the non-geometrical similarity and the coefficient of oxygen transfer (KLa) was kept constant between scales. The effectiveness of the new stirrer was validated in a pilot-scale in the presence of the cultures of bacteria, yeasts and filamentous fungi. Besides the studies on the stirrer, a process in a liquid medium of filamentous fungi was developed. Thus, the fermentation medium and the operating conditions of each steps of the fermentation (germination, vegetative growth, and sporulation) were optimized. This new process makes possible to obtain up to 200.106 cells per milliliter of culture on both laboratory and pilot scale (900L). Finally, the data gathered on the pilot scale was used to extrapolate this process to the industrial-scale of 10m3, Les objectifs de cette thèse sont, d’une part, de développer un système d’agitation performant en termes de transfert d‘oxygène et de forces de cisaillement afin qu’il soit adapté aux cultures de levures, de bactéries et de champignons filamenteux. D’autre part, de mettre au point un procédé de production des spores de champignons en milieu liquide pour remplacer la culture sur milieu solide (boîte de roux) utilisée au démarrage de la thèse par la société DSM. Ces travaux ont permis de sélectionner le mobile type « oreilles d’éléphant » comme système d’agitation. Son efficacité de transfert d’oxygène a été caractérisée dans une large gamme de débits d’air et de vitesses d’agitation. Les données obtenues ont été modélisées pour développer un outil de prédiction du KLa et d’extrapolation de procédé d’agitation. L’approche utilisée pour l’extrapolation est basée sur la similitude non géométrique et le maintien du coefficient de transfert d’oxygène (KLa) constant entre les échelles. L’efficacité du nouveau mobile d’agitation a été validée à l’échelle pilote en présence des cultures de bactéries, de levures et de champignons. Parallèlement au choix du mobile d’agitation, un procédé de production de spores de champignons filamenteux en milieu liquide a été développé. Ainsi, le milieu de culture et les conditions opératoires de chaque étape de la fermentation (germination, croissance végétative et sporulation) ont été optimisés. Ce nouveau procédé permet d’obtenir jusqu’à 200.106 cellules par millilitre de culture aussi bien à l’échelle du laboratoire que pilote (900L). Il a été aussi validé en présence de cultures de levures et de bactéries. Sur le même principe, les données obtenues à l’échelle pilote ont permis de développer un outil d’extrapolation à l’échelle industrielle de 10 m3

High throughput DNA sequencing technologies are fuelling an accelerating trend to assemble genomes de novo or to complete unfinished assemblies of previously sequenced genomes. Unfortunately, common DNA sequencing technology is limited to reading stretches of a few hundreds or thousands of base pairs only. Therefore, computational methods are needed to assemble entire genomes from large numbers of short DNA strands. However, standard algorithms that piece together DNA strands with overlapping sequences face important limitations due, for example, to regions of repeated sequences, thus leaving many genome assemblies incomplete (Alkan et al., 2011 [2]).We set out to develop a new methodology for genome assembly that promises to address some of these limitations. The method is based on Hi-C, a recent biochemical technique initially developed to analyse the 3D architecture of genomes (Lieberman-Aiden et al., 2009 [78]). In Hi-C experiments, DNA is crosslinked, cut by restriction enzymes, then diluted and religated. In standard Hi-C studies, a previously assembled genome is used to identify chimeric sequences among the ligation products, and map them to pairs of chromosomal loci, thereby yielding a genome-wide matrix of contact frequencies (Cournac et al., 2012 [27]). Our method essentially reverses this approach: Hi-C data are used to test for the physical continuity of the chromatin fibre as expected from a set of DNA segments (representing either a complete or incomplete chromosomal set). Physical-interactions aberrations in the contact matrix reveal structural incongruity, and lead to the reordering of chromosomal segments with respect to the physical properties and continuity of the fibre. This procedure improves genome assembly and/or identification of structural variants in re-sequenced genomes. Our approach uses a probabilistic (Bayesian) framework that assigns probabilities to different assemblies based on the experimental Hi-C data and on laws describing the physical properties of chromosomes (Wong et al. [146]). We will explain the methodology and the developed algorithms and provide results of applications to simulated and real Hi-C data from mutant and natural structural variants of yeast and fungi (Marie-Nelly et al., in prep). We also have developed algorithm that allow us to identify functional sequences in genomes from genomewide contact matrices. Notably, we annotated the centromeric position of the Naumovozyma castellii, an intriguing RNAi-containing yeast where centromere positions could not be determined with standard techniques (Marie-Nelly et al., submitted); Les technologies de séquençage de l'ADN à haut débit alimentent une tendance croissante à l'assemblage de génomes de novo ou à l'achèvement d'assemblages inachevés de génomes déjà séquencés. Malheureusement, la technologie standard de séquençage de l'ADN est limitée à la lecture de tronçons de quelques centaines ou milliers de paires de bases seulement. Par conséquent, des méthodes informatiques sont nécessaires pour assembler des génomes entiers à partir d'un grand nombre de brins d'ADN courts. Cependant, les algorithmes standard qui assemblent des brins d'ADN dont les séquences se chevauchent se heurtent à d'importantes limitations dues, par exemple, aux régions de séquences répétées, ce qui laisse de nombreux assemblages de génomes incomplets (Alkan et al., 2011 [2]).Nous avons entrepris de développer une nouvelle méthodologie pour l'assemblage de génomes qui promet de répondre à certaines de ces limitations. La méthode est basée sur Hi-C, une technique biochimique récente initialement développée pour analyser l'architecture 3D des génomes (Lieberman-Aiden et al., 2009 [78]). Dans les expériences Hi-C, l'ADN est réticulé, coupé par des enzymes de restriction, puis dilué et religé. Dans les études Hi-C standard, un génome préalablement assemblé est utilisé pour identifier les séquences chimériques parmi les produits de ligature, et les cartographier à des paires de loci chromosomiques, produisant ainsi une matrice de fréquences de contact à l'échelle du génome (Cournac et al., 2012 [27]). Notre méthode inverse essentiellement cette approche : Les données Hi-C sont utilisées pour tester la continuité physique de la fibre chromatinienne telle qu'attendue à partir d'un ensemble de segments d'ADN (représentant un ensemble chromosomique complet ou incomplet). Les aberrations des interactions physiques dans la matrice de contact révèlent une incongruité structurelle, et conduisent à la réorganisation des segments chromosomiques par rapport aux propriétés physiques et à la continuité de la fibre. Cette procédure améliore l'assemblage des génomes et/ou l'identification des variants structurels dans les génomes reséquencés. Notre approche utilise un cadre probabiliste (bayésien) qui attribue des probabilités aux différents assemblages en se basant sur les données expérimentales Hi-C et sur des lois décrivant les propriétés physiques des chromosomes (Wong et al. [146]). Nous expliquerons la méthodologie et les algorithmes développés et fournirons des résultats d'applications à des données Hi-C simulées et réelles provenant de mutants et de variantes structurelles naturelles de levures et de champignons (Marie-Nelly et al., en préparation). Nous avons également développé un algorithme qui nous permet d'identifier des séquences fonctionnelles dans les génomes à partir de matrices de contact à l'échelle du génome. Notamment, nous avons annoté la position centromérique de la Naumovozyma castellii, une levure intrigante contenant de l'ARNi où la position des centromères n'a pas pu être déterminée avec les techniques standard (Marie-Nelly et al., soumis).

Les sols forestiers sont des habitats hétérogènes, véritables réservoirs de microorganismes. Parmi ces microorganismes, les eucaryotes filamenteux (champignons et oomycètes) sont très divers et jouent un rôle important dans le fonctionnement et la durabilité des écosystèmes forestiers. Leur diversité et leur répartition spatiale à différentes échelles sont encore peu connues et les facteurs qui sous-tendent cette dispersion sont encore peu étudiés. Aussi, les objectifs étaient (i) d'exploiter le séquençage haut-débit pour des études d'écologie microbienne à large échelle et valider son application aux communautés d'oomycètes pathogènes en milieu forestier, (ii) de décrire ces communautés microbiennes, en termes de diversité et de structure, à différentes échelles spatiales (locale, régionale et continentale), (iii) de caractériser les variables biotiques et abiotiques structurant ces communautés et (iv) d'évaluer la réponse éventuelle des communautés aux variations climatiques. Une première étude pilote à l'échelle de la parcelle a été suivi de deux études à grande échelle spatiale le long de gradients environnementaux. Des gradients d'altitude et un gradient latitudinal, à l'échelle continentale, ont été utilisés comme gradient climatique. L'étude préliminaire a donc validé l'utilisation du pyroséquençage pour les communautés fongiques, et en particulier pour les espèces ectomycorhiziennes, et apporté des éléments pour établir une méthodologie d'échantillonnage couplée à cette technique. L'application de ces outils moléculaires à l'étude des communautés oomycètes pathogènes reste à optimiser. Les résultats obtenus sur les communautés fongiques telluriques suggèrent que dans l'hypothèse d'un réchauffement climatique, la richesse fongique ne serait pas directement affectée mais la composition des communautés le serait. La composition des communautés fongiques est également fortement liée au pH du sol. Ces résultats sont à affiner en étudiant plus en détail divers groupes taxonomiques et écologiques en lien avec des variables climatiques plus précises. Par ailleurs, de nombreuses perspectives sont envisageables pour améliorer la détection des oomycètes dans les sols forestiers, qui reste un challenge en écologie microbienne, Forest soils are heterogeneous habitats full of microorganisms. In particular, the filamentous eukaryotes (fungi and oomycetes) exhibit a huge diversity and play essential functions in the dynamic and sustainable growth of the forest ecosystem. However, their diversity and their distribution are poorly known; thus, so are the structuring factors of these microbial communities. The main goals were: (i) use a high-throughput pyrosequencing to study soil microbial communities at a broad geographical scale, and particularly to validate its use for the study of soil forest pathogenic oomycete communities, (ii) study the diversity and structure of fungal and pathogenic oomycetes communities at several spatial scales, (iii) identify possible climatic and edaphic variables structuring these communities and (iv) estimate the possible response of these microbial communities to climatic variation. A pilot study was undertaken at the stand scale. Then, two additional studies were carried out along environmental gradients at the regional and continental scales. The use of the pyrosequencing technique was found appropriate for the fungal communities, but difficulties arose in studying the pathogenic oomycete community. At the stand scale, results suggested the soil to be a valuable substitute for the roots to access the ectomycorrhizal richness and composition using pyrosequencing. The results along the broad scale gradients suggested that fungal richness may not be affected by climate warming but that the composition would be. Moreover, our work indicated that soil pH is a major factor explaining fungal community composition. The main conclusions are still to be confirmed and deeper knowledge of the response of different fungal phylum, or family, would be required. The detection and thus the diversity estimation of the pathogenic oomycetes in forest soil remains a current challenge.

The filamentous fungus, Podospora anserina, has two mating-type idiomorphs, mat+ and mat-. The mat+ sequence contains one gene FPR1, while mat- contains three genes: FMR1, SMR1 and SMR2. The molecular function of SMR1 is unknown, FPR1, FMR1 and SMR2 encode transcriptional regulators which control the fertilization (intercellular recognition) and the transition from a syncytium to a specialized dikaryotic hypha which contains one mat+ and one mat- nucleus (internuclear recognition). No exhaustive analysis is available for the genes involved in the intercellular recognition, while the mechanism of the internuclear recognition is unknown. In order to understand the mechanism of these events and to identify the target genes of mating-type transcription factors, we used a microarray approach. The transcriptomic profiles of the mat+ and mat- strains that are competent for fertilization revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1- and fpr1- mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat- strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. A comparison with similar data set from the two ascomycetes, Gibberella moniliformis and Sordaria macrospora, reveals significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species, questioning the function of these target genes. Internuclear recognition was investigated by the transcriptomic analysis of perithecia from two crosses expressing mat+ and mat- genes, respectively. The tow internuclear recognition models: nuclear identity and random segregation, were used to interpret our results. According to the former model, 27 target genes have been identified, while 154 target genes were identified with the latter model. A total of 46 mutant strains were constructed. However, these strains showed no defects in sexual development. Besides this microarray experiences, we have invalidated all HMG-box genes of P. anserina. The results show that the HMG-box genes have a very important role in sexual development, especially Pa_1_13940 which encodes the first identified regulator of Pezizomycotinan mating-type genes.; Le champignon filamenteux, Podospora anserina, possède deux types sexuels, mat+ et mat-, caractérisés chacun par une séquence spécifique. La séquence mat+ contient un seul gène FPR1; la séquence mat- contient trois gènes : FMR1, SMR1 et SMR2. La fonction moléculaire de SMR1 est inconnue, les autres gènes codent des facteurs de transcription qui contrôlent la fécondation (reconnaissance intercellulaire), et le passage d’un syncytium à un hyphe spécialisé binucléé contenant un noyau mat+ et un noyau mat- (reconnaissance internucléaire). Il n’y a pas eu d’analyse exhaustive des gènes impliqués dans la reconnaissance intercellulaire et le mécanisme de la reconnaissance internucléaire est encore inconnu. Afin de déterminer les cibles de FPR1 et FMR1, et les différents mécanismes impliqués, nous avons utilisé une approche microarray. Le profil transcriptomique des souches mat+ et mat- compétentes pour la fécondation a permis d’identifier 157 gènes cibles, et l’analyse transcriptomique des souches mutantes fpr1- et fmr1- a révélé que ces cibles peuvent être soit réprimées, soit activées par FMR1 ou FPR1, ou être sous le contrôle de ces deux facteurs. Ces expériences ont aussi détecté l’existence de 10 gènes activés ou réprimés au même niveau dans mat+ et mat-. La délétion de 32 gènes choisis parmi ces 167 gènes cibles n’a permis de mettre en évidence que deux gènes impliqués dans la fécondation. Les comparaisons des gènes cibles des facteurs de transcription MAT de Gibberella moniliformis et Sordaria macrospora avec ceux de P. anserina révèlent un nombre significatif de gènes cibles communs entre ces espèces, mais ces gènes ont des profils transcriptomiques différents, soulevant la question du rôle de ces gènes cibles. La recherche des gènes cibles de FPR1, FMR1 et SMR2 impliqués dans la reconnaissance internuléaire a été effectuée en comparant le transcriptome des périthèces issus de deux croisements, l’un n’exprimant que les gènes spécifiques mat+, l’autre que les gènes spécifiques mat-. Les résultats ont été interprétés selon le modèle d’identité nucléaire et le modèle de ségrégation aléatoire. Le premier modèle a conduit à l’identification de 27 gènes cibles, tandis que 154 gènes cibles ont été identifiés en appliquant le deuxième modèle. Au total 46 souches mutantes ont été construites. Cependant aucune délétion n’a affecté le développement sexué. En parallèle de ces expériences transcriptomiques, nous avons invalidé tous les gènes à HMG-box de P. anserina. Les résultats montrent que ces derniers ont un rôle très important dans le développement sexué, particulièrement Pa_1_13940 qui code un régulateur des gènes des types sexuels, le premier identifié chez les Pezizomycotina.

The filamentous fungus, Podospora anserina, has two mating-type idiomorphs, mat+ and mat-. The mat+ sequence contains one gene FPR1, while mat- contains three genes: FMR1, SMR1 and SMR2. The molecular function of SMR1 is unknown, FPR1, FMR1 and SMR2 encode transcriptional regulators which control the fertilization (intercellular recognition) and the transition from a syncytium to a specialized dikaryotic hypha which contains one mat+ and one mat- nucleus (internuclear recognition). No exhaustive analysis is available for the genes involved in the intercellular recognition, while the mechanism of the internuclear recognition is unknown. In order to understand the mechanism of these events and to identify the target genes of mating-type transcription factors, we used a microarray approach. The transcriptomic profiles of the mat+ and mat- strains that are competent for fertilization revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1- and fpr1- mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat- strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. A comparison with similar data set from the two ascomycetes, Gibberella moniliformis and Sordaria macrospora, reveals significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species, questioning the function of these target genes. Internuclear recognition was investigated by the transcriptomic analysis of perithecia from two crosses expressing mat+ and mat- genes, respectively. The tow internuclear recognition models: nuclear identity and random segregation, were used to interpret our results. According to the former model, 27 target genes have been identified, while 154 target genes were identified with the latter model. A total of 46 mutant strains were constructed. However, these strains showed no defects in sexual development. Besides this microarray experiences, we have invalidated all HMG-box genes of P. anserina. The results show that the HMG-box genes have a very important role in sexual development, especially Pa_1_13940 which encodes the first identified regulator of Pezizomycotinan mating-type genes., Le champignon filamenteux, Podospora anserina, possède deux types sexuels, mat+ et mat-, caractérisés chacun par une séquence spécifique. La séquence mat+ contient un seul gène FPR1; la séquence mat- contient trois gènes : FMR1, SMR1 et SMR2. La fonction moléculaire de SMR1 est inconnue, les autres gènes codent des facteurs de transcription qui contrôlent la fécondation (reconnaissance intercellulaire), et le passage d’un syncytium à un hyphe spécialisé binucléé contenant un noyau mat+ et un noyau mat- (reconnaissance internucléaire). Il n’y a pas eu d’analyse exhaustive des gènes impliqués dans la reconnaissance intercellulaire et le mécanisme de la reconnaissance internucléaire est encore inconnu. Afin de déterminer les cibles de FPR1 et FMR1, et les différents mécanismes impliqués, nous avons utilisé une approche microarray. Le profil transcriptomique des souches mat+ et mat- compétentes pour la fécondation a permis d’identifier 157 gènes cibles, et l’analyse transcriptomique des souches mutantes fpr1- et fmr1- a révélé que ces cibles peuvent être soit réprimées, soit activées par FMR1 ou FPR1, ou être sous le contrôle de ces deux facteurs. Ces expériences ont aussi détecté l’existence de 10 gènes activés ou réprimés au même niveau dans mat+ et mat-. La délétion de 32 gènes choisis parmi ces 167 gènes cibles n’a permis de mettre en évidence que deux gènes impliqués dans la fécondation. Les comparaisons des gènes cibles des facteurs de transcription MAT de Gibberella moniliformis et Sordaria macrospora avec ceux de P. anserina révèlent un nombre significatif de gènes cibles communs entre ces espèces, mais ces gènes ont des profils transcriptomiques différents, soulevant la question du rôle de ces gènes cibles. La recherche des gènes cibles de FPR1, FMR1 et SMR2 impliqués dans la reconnaissance internuléaire a été effectuée en comparant le transcriptome des périthèces issus de deux croisements, l’un n’exprimant que les gènes spécifiques mat+, l’autre que les gènes spécifiques mat-. Les résultats ont été interprétés selon le modèle d’identité nucléaire et le modèle de ségrégation aléatoire. Le premier modèle a conduit à l’identification de 27 gènes cibles, tandis que 154 gènes cibles ont été identifiés en appliquant le deuxième modèle. Au total 46 souches mutantes ont été construites. Cependant aucune délétion n’a affecté le développement sexué. En parallèle de ces expériences transcriptomiques, nous avons invalidé tous les gènes à HMG-box de P. anserina. Les résultats montrent que ces derniers ont un rôle très important dans le développement sexué, particulièrement Pa_1_13940 qui code un régulateur des gènes des types sexuels, le premier identifié chez les Pezizomycotina.

Background Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation. Results In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases. Conclusion This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2 . Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex] 12 and [Hex] 8 , respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.

Background The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection. Results After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from T. reesei. Conclusions Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus T. reesei gives new insights into the physicochemical parameters and biodiversity in this family.

Studies on ensiling of spent solids from solid state fermentation process for production of cellulases by #Trichoderma harzianum$ showed that good quality ensiled solids can be obtained by using about 43% initial substrate dry matter with 0.3% ensiling additive. (Résumé d'auteur)

Abstract Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.

International audience; Background: Biological methods based on enzymatic activity measurements may offer promising perspectives for ecotoxicological risk assessment in soils contaminated by heavy metals. Because of their ecological pertinence, we retain filamentous fungi as models for the development of enzymatic methods. Aims: The present study aims to assess both selectivity and sensitivity of the response of exocellular oxidases secreted by the basidiomycete Trametes versicolor exposed to metals. Methods: We spiked with high amounts of metals pure cultures performed in liquid media to assess the selectivity of the enzymatic response. We used media exhibiting several levels of metal complexation to measure its sensitivity. Results: Laccase and manganese-peroxidase activities were highly stimulated in the presence of copper, mercury and cadmium. By contrast, lead and zinc were without significant effects on these activities. Lignin-peroxidase activity was not measured in the control cultures, but it was strongly increased after fungal exposure to copper. Assays performed with mixtures of metals, reflecting soil multiple contaminations, provided contrasted results. Then, culture media exhibiting low complexing properties led to an increased bioavailability of metals. As a consequence, we were able to measure the enzymatic responses at environmental levels of contaminants. Conclusion: Our results validate our hypothesis and allow us to develop studies in soils. We will now introduce Trametes versicolor in contaminated soils and measure the resulting enzymatic activities.

Mushrooms to restore polluted soil

International audience; Un recensement national des sites pollués réalisé en 1994 par le ministère de l'Environnement a répertorié 669 sites contaminés, qui se répartissent en trois grands types : les anciennes décharges, qui ont été utilisées sans respecter les règles techniques actuelles et dont le sous-sol inadapté a conduit à une pollution des eaux, les dépôts de déchets, occasionnés par des faillites d'entreprises ou des pratiques frauduleuses d'importation ou d'élimination de déchets et, enfin, les sols pollués par des retombées, des infiltrations ou des déversements de substances polluantes, liés à l'exploitation d'une installation industrielle ou à un accident de transport.Cet inventaire sera réactualisé fin 1996. Cependant, on se heurte toujours à l'absence de normes fran- çaises aussi bien sur les teneurs maximales admises pour les composés organiques dans les sols que sur les techniques d'analyse permettant d'évaluer ces teneurs. Les sites pollués constituent pourtant un risque à moyen terme pour les eaux de surface et souterraines et pour l'usage même des sols (habitat, cultures, implantation d'activités).La diversité chimique apparente de la pollution d'un site est généralement faible et se limite, pour les polluants organiques, à quelques molécules (appartenant à une ou plusieurs familles chimiques) produites par l'activité industrielle exercée sur le site. En France, les hydrocarbures aromatiques polycycliques (HAP) et les polychlorobyphényles (PCB) sont les plus fréquemment rencontrés. Pour les HAP, plus de 400 sites sont recensés. Sur un même site, les teneurs en ces molécules sont très hétérogènes et elles peuvent atteindre localement 1 000 à 10 000 ppm (mg/kg). Certains sites accueillant des transformateurs électriques contiennent des matériaux ou des sols pollués par les PCB à des teneurs de quelques centaines de ppm. On trouve également parmi les polluants organiques des produits pétroliers, des munitions, des explosifs, des produits à usage agro-pharmaceutique. Les contaminations peuvent survenir sur les lieux de production ou sur ceux de stockage.

L'objectif de ce travail est d'établir les connaissances qui permettront la mise au point des méthodes permettant de conserver par ensilage de la pulpe de café décaféinée. Pour cela, nous avons utilisé des ferments mixtes de bactéries lactiques et de champignons filamenteux cultivés en fermentation solide anaérobie ou aérobie. La pulpe de café est quantitativement et qualitativement le sous-produit le plus important du traitement par voie humide des cerises de café. Ce résidu humide et saisonnier engendre de sérieux problèmes de pollution qu'il serait possible de résoudre en le stabilisant par ensilage. De part sa composition chimique, la pulpe de café pourrait être utilisée en alimentation animale. Mais il faut alors éliminer les composés toxiques que sont la caféine et les composés phénoliques. Des études préalables ont démontré que des champignons filamenteux sont capables de dégrader la caféine en FMS. Mais au préalable à toute étude de conservation ou de valorisation de ce substrat, nous avons identifié et quantifié la microflore endogène de la pulpe. Celle-ci varie de 700 000 à 112 000 000 UFC/g PMS suivant la variété du café, l'origine géographique et le type de traitement appliqué aux cerises de café. La microflore lactique endogène de la pulpe de café (10 000 UFC/g PMS) s'exprime dès que les conditions environnementales (anaérobiose et humidité) sont favorables. Nous avons d'autre part testé l'effet de l'apport de différents ferments lactiques. La qualité des ensilages est parfaitement acceptable. Quelque soit le type d'ensilage réalisé, la caféine de la pulpe de café présente à la mise en silos, n'est pas dégradée. Après un criblage en FMS de huit souches de champignons filamenteux, nous avons continué notre étude avec #Penicillium$ V33A25. Ce microorganisme dégrade la caféine de la pulpe de café à 94 % après 30 heures de fermentation aérobie, avant la phase de sporulation. (Résumé d'auteur)

We have examined two A. Niger esterases (FAE-III (ferulic acid esterase III) and CinnAE (cinnamic acid esterase)) for their ability to release ferulic acid from maize bran, a particularly rich source of ferulic acid. However, even though both enzymes exhibited significant activites on novel feruloylated oligosaccharides derived from maize bran (including FAXX (O-(5-O-feruloyl-α-l-arabinofuranosyl)-(1→3)-O-β-xylopyranosyl-(1→4)-d-xylopyranose)), neither esterase was efficient in removing ferulic acid from the whole bran, even in the presence of other carbohydrases. It is shown that the kinetics of ferulic acid release from feruloylated oligosaccharides was influenced by (a) the nature of the sugar-sugar linkage, and (b) the type of sugar present. The results suggest that enzymic release of ferulic acid from maize bran is limited by physical and steric factors, not by the chemical nature of the linkage.

Recovery of cellulases from solid state culture of #Trichoderma harzianum$ was efficiently achieved by hydraulic pressing. Pressing of fermented solids yielded carboxymethyl-cellulase (CMCase) extraction efficiency of 71% and a ratio of leachate to fermented solids of 0.58 (v/w). Addition of water to pressed solids and second pressing improved the efficiency (95%) with simultaneous increase in the ratio to 1.16 (v/w). The overall extraction of filter paper activity was lower (85%) than that of CMCase. This technique is simple and its extraction efficiency is similar to that obtained in multiple-contact counter-current systems. The hydraulic press in its individuality was not used earlier to leach the product from fermented solids. (Résumé d'auteur)

ISBN 90-247-3153-4 89 ref.; International audience

International audience