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Sterilization is essential for discriminating biotic responses from abiotic reactions in laboratory experiments investigating biogeochemical processes of complex natural samples. However, the conventional methods used to effectively sterilize materials or culture media do not allow sterilizing complex natural samples while maintaining biogeochemical balances. The aim of this study was to develop a low-cost and easy-to-use method to obtain geochemically unmodified and sterilized samples from complex lacustrine or coastal marine ecosystems. In preliminary assays, the impact of several sterilization methods (autoclaving, chemical poisoning, microwave, UV irradiation) on the trace metals balances was studied using borosilicate glass (BG), fluorinated ethylene-propylene (FEP) or polyethylene terephthalate (PET) bottles. Unlike other methods, UV sterilization had minor effects on the distribution of dissolved trace metals. Additional tests using complex lacustrine and coastal marine samples under 10 g/L sediments were performed using a homemade UV sterilization chamber designed to simultaneously irradiate a large number samples. Results showed: • very reproducible UV tests in BG and FEP bottles • faster sterilization using FEP bottles than using BG bottles • low variations of dissolved trace metals concentrations, except for Al, Cu, Fe and Zn Method name: UV sterilization of complex natural (lacustrine or coastal marine) samples, Keywords: UV, Sterilization, Trace metals, Natural ecosystems, Complex matrices

International audience; Anaerobic biotechnology using sulfate-reducing bacteria (SRB) is a promising alternative for reducing long-term stockpiling of phosphogypsum (PG), an acidic (pH ∼3) by-product of the phosphate fertilizer industries containing high amounts of sulfate. The main objective of this study was to evaluate, for the first time, the diversity and ability of anaerobic marine microorganisms to convert sulfate from PG into sulfide, in order to look for marine SRB of biotechnological interest. A series of sulfate-reducing enrichment cultures were performed using different electron donors (i.e., acetate, formate, or lactate) and sulfate sources (i.e., sodium sulfate or PG) as electron acceptors. Significant sulfide production was observed from enrichment cultures inoculated with marine sediments, collected near the effluent discharge point of a Tunisian fertilizer industry (Sfax, Tunisia). Sulfate sources impacted sulfide production rates from marine sediments as well as the diversity of SRB species belonging to Deltaproteobacteria. When PG was used as sulfate source, Desulfovibrio species dominated microbial communities of marine sediments, while Desulfobacter species were mainly detected using sodium sulfate. Sulfide production was also affected depending on the electron donor used, with the highest production obtained using formate. In contrast, low sulfide production (acetate-containing cultures) was associated with an increase in the population of Firmicutes. These results suggested that marine Desulfovibrio species, to be further isolated, are potential candidates for bioremediation of PG by immobilizing metals and metalloids thanks to sulfide production by these SRB.

International audience; High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (∼11), low-temperature (

This study aimed at evaluating potential arsenic (As) mobility in an industrially contaminated soil (64mg/kg of As) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobic conditions using a laboratory batch experiment. In the presence of exogenous carbon sources, As-V initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides were solubilized and mainly reduced to As-III by indigenous soil microflora. After a 1-month incubation period in these biotic conditions, As-III accounted for 80-85% of the total dissolved As and more than 60% of the solid-phase As. Bacterial community structure (i.e., 16S rDNA-based capillary electrophoresis single-strand conformation polymorphism profiles) changed with incubation time and As amendment. The detection of distantly related arsenate respiratory reductase genes (arrA), as functional markers of As-V respirers, indicates that novel dissimilatory As-V-reducing bacteria may be involved in As biotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, a crucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible for heterotrophic As-V reduction to As-III, which may increase As toxicity and mobility in the contaminated soils.

International audience; This paper reviews and discusses the impact of byproducts released during pretreatment of lignocellulosic materials on anaerobic mixed cultures producing hydrogen and methane. 5-HMF, furfural, phenolic compounds and aldehydes, are strong inhibitors of biohydrogen production but can be further converted into methane. This finding can be explained by differences in both process parameters: anaerobic digestion is performed with more complex mixed cultures, lower substrate/inoculum or byproducts/inoculum ratios and longer batch incubation times than dark fermentation. Indeed, the presence of byproducts may require an adaptation phase of the microbial community leading to longer lag phase in dark fermentation. Finally, the presence of pretreatment by-products may lead to a metabolic shift from hydrogen production to no H2-producing ethanol and lactate pathways and whatever the route of dark fermentation, metabolites can be all further converted into methane, at different rates.

National audience; Ces travaux ont été réalisés en utilisant un cocktail enzymatique mis au point par Protéus. Ce cocktail a été utilisé aussi bien pour le prétraitement de la voie fermentaire (procédé en deux étapes : hydrolyse puis fermentation sombre) mais aussi, et c’est un point original, en addition directe dans le fermenteur (procédé en une étape). Certains résultats de ces travaux ont été publiés dans le journal scientifique International Journal of Hydrogen Energy*. Quels que soient le procédé (1 ou 2 étapes) et le type de paille de blé utilisé (stérile ou non stérile), la production d’hydrogène par gramme de matière organique de paille est doublée grâce à l’addition du cocktail enzymatique de Protéus.

International audience; Fermentative biohydrogen technologies are environment-friendly ways for producing renewable hydrogen from a wide range of organic sources, and at a low cost. The main restriction of such bioprocesses remains their high instability when operated with mixed cultures. The role of minority bacteria has often been suspected to explain metabolic instability in hydrogen fermentation, but has never really been investigated. The trophic competition of lactic bacteria with hydrogen-producing bacteria for substrate is well documented in literature. The consequence is a loss of hydrogen production due to shift towards unwanted fermentative metabolisms. However, experimental studies on non-trophic interactions in engineered ecosystems are scarce. In this work, two complex hydrogen-producing microbial ecosystems deriving from anaerobic sludge treating industrial waste were used. The molecular characterization of mixed cultures was done using CE-SSP fingerprinting patterns. The first ecosystem (EcoA) presented a low hydrogen production (440 mL.H2.L-1) but was metabolically stable (CV=8%), and the second ecosystem (EcoB) produced more hydrogen (2157 mL.H2.L-1) but was metabolically unstable (CV=36%). The two ecosystems chosen corresponded to the lower and the upper range of hydrogen productivity found in the literature. The impact of a contamination with nine various exogenous bacterial strains (Clostridium acetobutylicum, Clostridium pasteurianum, Lactobacillus bulgaris, Enterobacter cloacae, Escherichia coli, Enterococcus casseliflavus, Desulfovibrio vulgaris, Pseudomonas fluorescens, Ralstonia eutropha) exhibiting distinct metabolic abilities and belonging to diverse genera was investigated. In EcoA, a positive effect on the hydrogen yield was observed after addition of two hydrogen-producing facultative anaerobic bacteria, Escherichia coli (1.5-fold increase, p=0.05) and Enterobacter cloacae (2.5-fold increase, p=0.04). Interestingly, Ralstonia eutropha led to a four-fold increase in hydrogen production (p

International audience; The three As(III)-oxidizing Betaproteobacteria Thiomonas delicata, Thiomonas cuprina and "Thiomonas arsenivorans" were isolated from mining sites in geographically distinct areas, namely Japan, Germany and France, respectively. They are all able to oxidize As(III), but only "T. arsenivorans" and T. cuprina show efficient autotrophic growth with As(III) and are able to grow on single carbon compounds. Those two organisms are motile, but T. delicata is not. Only T. cuprina can grow autotrophically on chalcopyrite. The three strains share more than 99% gene sequence similarities for their 16S rRNA genes and their 16S-23S ITS regions. DNA-DNA hybridization results are above or close to the suggested threshold value of 70% for the definition of bacterial species. They show very similar fatty acids profiles with differences only in five minor ones. They possess phylogenetic and chemotaxonomic similarities supporting their reclassification as a single species. We propose to reassign "T. arsenivorans" and T. cuprina as strains of T. delicata (type strain DSM 17897T).

International audience; Hydrogen (H2) is one of the most promising energy carriers because it is energy dense, clean burning and can be renewably produced. Dark fermentation using mixed cultures is an attractive biotechnology to produce H2 at low cost from lignocellulosic biomass, the most abundant renewable resource. However, the dilute acid pre-treatment, generally used to convert the complex biomass into monosaccharides, also releases other degradation products which can inhibit microbial growth and fermentation. The aim of this study was to investigate the effects of added lignocellulose-derived compounds (i.e. furan derivatives such as furfural, HMF, phenolic compounds such as phenol, syringaldehyde, vanillin and kraft and organosolv lignin) on fermentative H2 production and associated bacterial communities. H2-producing batch tests were performed using anaerobic heat-treated digested sludge with various substrates: xylose alone as control, xylose in presence of inhibitory compounds and inhibitory compounds alone. Bacterial community were monitored by Capillary-Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) fingerprinting using bacterial-specific 16S rDNA primers and H2-producing bacterial-specific primers targeting functional hydA genes, encoding the catalytic sub-unit of [FeFe]-hydrogenases. Results indicated that all the lignocellulose-derived compounds tested were unable to support fermentative H2 production in the absence of additional carbon source. When added to xylose, all tested compounds significantly decreased fermentative H2 production performances. H2 yields were more affected by the addition of furan derivatives than phenol compounds. Except for lignins, the higher the molecular mass was, the shorter the lag phase time was. Noteworthily, lignins affected slightly the lag phase time but largely contributed to the H2 production instability, which can be explained by their physicochemical properties (e.g. hydrophobicity, ramification, methoxylation). Although positive correlation was observed between butyrate and H2 productions, the composition and distribution of soluble metabolites varied in accordance with the inhibitor type. Likewise, changes in bacterial community structure and diversity were also associated with inhibitor tested. Clostridial species dominated all mixed cultures and their key role in the H2 production was confirmed by the detection of clostridial hydA genes. Interestingly, Clostridium beijerinkii was found less sensitive to inhibitors, making it ideal candidate for H2 production from hydrolysates of lignocellulosic biomass. To improve the efficiency of fermentative H2 production from lignocellulosic biomass, these results suggest the application of efficient delignification and depolymerisation processes releasing low amounts of furan derivatives and phenolic compounds and/or the use of microbial communities adapted to lignocellulose-derived inhibitors.

International audience; To investigate the alkane-hydroxylating system of isolate SP2B, closely related to Rhodococcus ruber DSM 43338T and uncharacterized so far for its alkane degradation genes. Methods and Results: Although isolate SP2B and reference strain can grow on by-products from hexane degradation, the type strain R. ruber was unable, unlike SP2B isolate, to use short-chain alkanes, as assessed by gas chromatography. Using PCR with specific or degenerated primers, inverse PCR and Southern blot, two alkane hydroxylase encoding genes (alkB) were detected in both bacteria, which is in agreement with their alkane range. The first AlkB was related to Rhodococcus AlkB7 enzymes and contains a nonbulky residue at a specific position, suggesting it might be involved in medium- and long-chain alkane oxidation. The second partial alkB gene potentially belongs to alkB5-type, which was found in bacteria unable to use hexane. Moreover, a partial P450 cytochrome alkane hydroxylase, thought to be responsible for the hexane degradation, was detected only in the isolated strain. Conclusions : Rhodococcus ruber SP2B should prove to be a promising candidate for bioremediation studies of contaminated sites because of its large degradation range of alkanes. Significance and Impact of the Study : This is the first thorough study on R. ruber alkane degradation systems.