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International audience; This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY)The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the β-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ββ, SS βO4 and GG β5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

International audience; Biomass recalcitrance is one of the main bottlenecks in lignocellulosic biorefinery deployment. Physico-chemical pretreatments and enzymatic hydrolysis are two procedures that can be combined to overcome this recalcitrance. In this study microwave pretreatment has been selected for its relevant conditions that allow for biomass recalcitrance to be reduced, along with the maintenance of a low consumption of energy and reactants. A xylanolytic enzymatic cocktail, Rovabio® Advance, was investigated for its ability to hydrolyze maize stalks and Miscanthus leaves after pressurized, chemical-free microwave pretreatment. This combination was implemented to increase the breakage of ester bonds and thus facilitate the release of p-hydroxycinnamic acids. This study demonstrates how, in comparison with Miscanthus, both pretreatments are more effective in releasing p-hydroxycinnamic acids from maize stalks, due to their lower parietal content. The successive free-chemical process seems to be particularly promising on maize stalks, since it led to a ferulic acid release yield of 18.2%, compared to 5.5% for microwave pretreatment only or 7.6% when performing enzymatic hydrolysis without a microwave pretreatment step.

[Background] Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide., [Results] We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners., This work was supported by The French National Agency for Research (ANR-12-BIME-0009, ANR-14-CE06-0020-01). The work by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The work by CSIC was supported by the Spanish Ministry of Economy, Industry and Competitiveness (BIO2017-86559-R). The HPC resources of Aix-Marseille Université was supported by The French National Agency for Research (ANR-10-EQPX-29-01).

International audience; For the first time, Polyporus brumalis BRFM985 was cultivated on wheat straw to investigate the simultaneous effects of pretreatment parameters on anaerobic digestion: these include initial substrate humidity, temperature, duration, and metal supplementation. Surface response methodology was applied to quantify the importance of each parameter, as well as the synergistic effects between them. Firstly, metal addition and secondly, pretreatment duration, both resulted in a positive impact. According to calculations, the highest methane production (182 dm3 of methane per kilogram of initial Total Solids) is associated to pretreatment with metal addition during 20 days. In comparison with the least optimal conditions (118 dm3.kg−1 without metal addition, during 15 days), this result implies a 52% increase.

The purpose of this work was to optimize the pretreatment process of wheat straw by Polyporus brumalis_BRFM985 in order to improve carbohydrate accessibility for more efficient bioconversion. Indeed, there is growing demands to develop sustainable routes for lignocellulosic feedstocks valorization into value‐added products in energy, chemicals, materials, and animal feed fields. To be achieved, implementation of cheap and ecofriendly biomass pretreatment processes is necessary. In this frame, white rot basidiomycetes, well known for their ability to degrade lignin efficiently and selectively, are of great interest. The pretreatment of wheat straw by Polyporus brumalis_BRFM985 was performed in packed bed bioreactor and optimized using response surface methodology. The four pretreatment parameters optimized were metals addition (Cu, Mn, and Fe), time of culture, initial water content, and temperature. Multicriteria optimization highlighted that wheat straw pretreatment by Polyporus brumalis_ BRFM985 in the presence of metals with high initial water content of 3.6 g H2O/g at 27°C for 15–16 days led to an improvement of carbohydrate accessibility with minimal matter loss.

Directeur de l'ouvrage : Arnaud Diemer Directeur de l'ouvrage : Florian Diericks Directeur de l'ouvrage : Ganna Gladkykh Directeur de l'ouvrage : Manuel E Morales Directeur de l'ouvrage : Tim Parrique Directeur de l'ouvrage : Julian Torres Auteur de l'ouvrage : Arnaud Diemer Directeur de l'ouvrage : Arnaud Diemer Directeur de l'ouvrage : Florian Diericks Directeur de l'ouvrage : Ganna Gladkykh Directeur de l'ouvrage : Manuel E Morales Directeur de l'ouvrage : Tim Parrique Directeur de l'ouvrage : Julian Torres Auteur de l'ouvrage : Arnaud DiemerDirecteur de l'ouvrage : Arnaud DiemerDirecteur de l'ouvrage : Florian DiericksDirecteur de l'ouvrage : Ganna GladkykhDirecteur de l'ouvrage : Manuel E MoralesDirecteur de l'ouvrage : Tim ParriqueDirecteur de l'ouvrage : Julian TorresAuteur de l'ouvrage : Arnaud Diemer; Exploration of fungal biodiversity for the deconstruction of lignocellulosic biomass and the implementation of new biosynthetic pathways for green chemistry. European Union and Sustainable Development: Challenges and Prospects

Casein micelles were loaded with iron to create a dispersible delivery format for insoluble iron by exposing milk at chilled temperatures to a high concentration of soluble iron (up to 20 mmol kg−1 ferrous and ferric chloride). The loading was maximised by applying a pH-cycle to the fortified milk by means of carbonation. Upon acidification of fortified milk, no release of iron was observed, except at the highest concentration. Changes in the buffering capacity as a function of pH confirmed the formation of colloidal iron phosphates. Overall, most properties of the micelles did not change: hydration, protein distribution between soluble and colloidal phase remained constant, but zeta potential decreased slightly and curd formation upon renneting became much slower. The renneting behaviour could be improved by carbonation or storage at 30 °C for a day. Iron-fortified milk samples were stable under heating, except when fortification was achieved by means of 20 mmol kg−1 ferric chloride. The most obvious difference of iron-fortified milk is its appearance: samples fortified with ferrous chloride were darker than control, whereas samples fortified with ferric chloride were more red/yellow.

Physicochemical properties of non-fat, low heat, reconstituted skim milk subjected to pH cycling using CO2 as an acidifying agent were studied. The pH was dropped to 5.5 at 4 °C for 15 min and temperature was subsequently kept at 4 °C or increased to 20 and 30 °C for further 45 min, before degassing under vacuum. Even though many overall properties of the micelles were not affected (calcium and protein content, size, zeta potential, acid gelling properties), it was shown that the internal organisation of the micelles did change. The structure of the micelle was dependent on carbonation temperature and evolved during chilled storage, as indicated by the rennet clotting time and micelle hydration variations. In contrast, modifications of the micellar calcium phosphate salt form, as reflected in the buffering capacity curves, did not depend on temperature of carbonation.

International audience; Background: Lactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Yeasts have been engineered to produce high levels of lactic acid at low pH from glucose but not from carbohydrate polymers (e.g. cellulose, hemicellulose, starch). Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. However, they have never been used for lactic acid production. Results: To investigate the feasibility of lactic acid production with Aspergillus, the NAD-dependent lactate dehydrogenase (LDH) responsible for lactic acid production by Rhizopus oryzae was produced in Aspergillus brasiliensis BRFM103. Among transformants, the best lactic acid producer, A. brasiliensis BRFM1877, integrated 6 ldhA gene copies, and intracellular LDH activity was 9.2 x 10(-2) U/mg. At a final pH of 1.6, lactic acid titer reached 13.1 g/L (conversion yield: 26%, w/w) at 138 h in glucose-ammonium medium. This extreme pH drop was subsequently prevented by switching nitrogen source from ammonium sulfate to Na-nitrate, leading to a final pH of 3 and a lactic acid titer of 17.7 g/L (conversion yield: 47%, w/w) at 90 h of culture. Final titer was further improved to 32.2 g/L of lactic acid (conversion yield: 44%, w/w) by adding 20 g/L glucose to the culture medium at 96 h. This strain was ultimately able to produce lactic acid from xylose, arabinose, starch and xylan. Conclusion: We obtained the first Aspergillus strains able to produce large amounts of lactic acid by inserting recombinant ldhA genes from R. oryzae into a wild-type A. brasiliensis strain. pH regulation failed to significantly increase lactic acid production, but switching nitrogen source and changing culture feed enabled a 1.8-fold increase in conversion yields. The strain produced lactic acid from plant biomass. Our findings make A. brasiliensis a strong contender microorganism for low-pH acid production from various complex substrates, especially hemicellulose.

The potential of fungal pretreatment to improve fermentable sugar yields from wheat straw or Miscanthus was investigated. We assessed 63 fungal strains including 53 white-rot and 10 brown-rot fungi belonging to the Basidiomycota phylum in an original 12 day small-scale solid-state fermentation (SSF) experiment using 24-well plates. This method offers the convenience of one-pot processing of samples from SSF to enzymatic hydrolysis. The comparison of the lignocellulolytic activity profiles of white-rot fungi and brown-rot fungi showed different behaviours. The hierarchical clustering according to glucose and reducing sugars released from each biomass after 72 h enzymatic hydrolysis splits the set of fungal strains into three groups: efficient, no-effect and detrimental-effect species. The efficient group contained 17 species belonging to seven white-rot genera and one brown-rot genus. The yield of sugar released increased significantly (max. 62%) compared with non-inoculated controls for both substrates.

Background Filamentous fungi are well known for their ability to degrade lignocellulosic biomass and have a natural ability to convert certain products of biomass degradation, for example glucose, into various organic acids. Organic acids are suggested to give a competitive advantage to filamentous fungi over other organisms by decreasing the ambient pH. They also have an impact on the ecosystem by enhancing weathering and metal detoxification. Commercially, organic acids can be used as chemical intermediates or as synthons for the production of biodegradable polymers which could replace petroleum-based or synthetic chemicals. One of the advantages of filamentous fungi as biotechnological production platforms for synthetic biology is their ability to degrade vegetal biomass, which is a promising feedstock for the biotechnological production of organic acids. The Fungal Culture Collection of the International Centre of Microbial Resources (CIRM-CF), curated by our laboratory, contains more than 1600 strains of filamentous fungi, mainly Basidiomycetes and Ascomycetes. The natural biodiversity found in this collection is wide, with strains collected from around the world in different climatic conditions. This collection is mainly studied to unravel the arsenal of secreted lignocellulolytic enzymes available to the fungi in order to enhance biomass degradation. While the fungal biodiversity is a tremendous reservoir for “green” molecules production, its potentiality for organic acids production is not completely known. Results In this study, we screened 40 strains of Ascomycota and 26 strains of Basidiomycota, representing the distribution of fungal diversity of the CIRM-CF collection, in order to evaluate their potential for organic acid and ethanol production, in a glucose liquid medium. We observed that most of the filamentous fungi are able to grow and acidify the medium. We were also able to discriminate two groups of filamentous fungi considering their organic acid production at day 6 of incubation. This first group represented fungi co-producing a wide variety of organic acids and ethanol at concentrations up to 4 g.L−1 and was composed of all the Aspergilli and only 3 other Ascomycota. The second group was composed of the remaining Ascomycota and all the Basidiomycota which produced mainly ethanol. Among the Basidiomycota, two strains produced oxalic acid and one strain produced gluconic and formic acid. Six strains of Aspergillus producing high concentrations of oxalic, citric and gluconic acids, and ethanol were selected for metabolism analysis. Conclusion These results illustrate the versatility in metabolites production among the fungal kingdom. Moreover, we found that some of the studied strains have good predispositions to produce valuable molecules. These strains could be of great interest in the study of metabolism and may represent new models for synthetic biology or consolidated bioprocessing of biomass. Electronic supplementary material The online version of this article (doi:10.1186/s40694-014-0001-z) contains supplementary material, which is available to authorized users.

The use of ion-exchange chromatography and 31 P nuclear magnetic resonance ( 31 P NMR) to analyse the composition and the chain length of phosphate emulsifying salts were studied, as well as the impact of these salts in dairy products. Ion chromatography was more appropriate than 31 P NMR to study polyphosphate composition in complex environments, whereas interactions between phosphate species and dairy components were elucidated by 31 P NMR. Phosphate species interacting with calcium, as well as the percentage of chelated calcium, were identified using 31 P NMR. Thus, ion chromatography and solid-sate 31 P NMR could be used as complementary methods to study compositions of polyphosphate blends and their interactions with dairy matrices.

International audience; Antimicrobial bio-sourced films based on poly(lactic acid) containing either carvacrol or allyl isothiocyanate (AITC) were prepared and their antimicrobial properties were assessed on Botrytis cinerea during storage and after a high pressure (HP) "pasteurisation-like" treatment (up to 800 MPa at ambient temperature). A dry process (extrusion + thermomoulding) was used to shape the material. The high temperature encountered during film processing dramatically decreased the carvacrol and AITC content in the film, leading to a less efficient antimicrobial activity. The use of beta-cyclodextrin (beta-CD) to encapsulate the active compounds before film processing proved to be efficient to protect the AITC against thermal degradation and to control its release from the films during its use. PLA-based films containing either AITC or beta-CD encapsulated AITC showed a significant activity against B. cinerea. An effective combination between the antimicrobial activity of AITC-based films and the high pressure treatment was observed on a model food system (PDA) inoculated with N x 10(4) (N similar to 1-9) conidia of B. cinerea. An HP treatment of only 300 MPa associated with an antimicrobial PLA/beta-CD system providing an initial quantity of active agents equivalent to 4 mg of AITC/L of air (i.e. almost 2 folds lower than the minimal inhibition concentration of the active packaging used alone, which was determined to be equal to 10 mg/L of air in the same conditions) was found more efficient (total inhibition of B. cinerea growth during 10 days) than an 800 MPa HP treatment used alone (increase of the lag phase growth of 3.3 days). Industrial relevance: The consumer demand for "fresh like" product containing reduced amount of preservatives without compromising human and environmental safety needs the development of new preservation strategies. As a consequence, the concept of "hurdle technologies" has risen up. The combined effect of HP treatment and volatile antimicrobial packaging allowed the use of lower individual treatment intensities to inhibit B. cinerea growth. Combining such "hurdles" is of relevance in the context of development of low-cost and eco-friendly food technologies.

International audience; Reconstituted skim milk was fortified at 2, 5, 10 and 20 mmol Fe kg(-1) with ferric and ferrous chloride iron with or without reversible acidification by injection of CO2 under pressure. Carbonation improved transfer of iron from the soluble to the colloidal phase and accelerated ferrous iron oxidation. Fe-57 Mossbauer spectra of the freeze-dried casein micelles collected by centrifugation demonstrated that iron is present in a distorted octahedral coordination and is chelated by phosphate rather than citrate. Milk iron fortification induced several changes in the mineral component of the casein micelles, which are explained by the formation of a tertiary complex: inorganic phosphate-iron-organic phosphate.

Correspondance: sraouche@univ-montp2.fr; International audience; Casein micelle stability and reactivity were assessed on milk subjected to reversible acidification by carbonation. Pressurised CO2 was injected at 4 degrees C, leading to controlled acidification from 6.63 +/- 0.02 to a target pH (5.5 or 5.2). After holding the pressurised milk under these conditions for 15 or 60 min, the pressure was released and the milk pH returned to its initial value under stirring and vacuum degassing. Upon CO, treatment, calcium and protein partition, zeta potential and size of casein micelles were restored directly after nentralisation. The rheological properties of the gel obtained by acid coagulation Of CO2-treated milk did not change as a result of carbonation. Micelle hydration increased after neutralisation and during storage. Milk buffering capacity in the pH range of 4.5-5.5 decreased after neutralisation of milk acidified by carbonation, but increased during chilled storage of this milk. Holding time of carbonated milk at low pH was found to have no impact on the physicochemical characteristics of casein micelles and the rheological properties of the gel obtained by acid coagulation of this milk

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