Multi-system Nernst–Michaelis–Menten model applied to bioanodes formed from sewage sludge

Bioanodes were formed under constant polarization at 0.2 V/SCE from fermented sewage sludge. Current densities reached were 9.3 ± 1.2 A m2 with the whole fermented sludge and 6.2 ± 0.9 A m2 with the fermented sludge supernatant. The bioanode kinetics was analysed by differentiating among the contributions of the three redox systems identified by voltammetry. Each system ensured reversible Nernstian electron transfer but around a different central potential. The global overpotential required to reach the maximum current plateau was not imposed by slow electron transfer rates but was due to the potential range covered by the different redox systems. The microbial communities of the three bioanodes were analysed by 16S rRNA gene pyrosequencing. They showed a significant microbial diversity around a core of Desulfuromonadales, the proportion of which was correlated with the electrochemical performance of the bioanodes

Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes

Biocathodes polarized at high potential are promising for enhancing Microbial Fuel Cell performances but the microbes and genes involved remain poorly documented. Here, two sets of five oxygen-reducing biocathodes were formed at two potentials (−0.4 V and +0.1 V vs. saturated calomel electrode) and analyzed combining electrochemical and metagenomic approaches. Slower start-up but higher current densities were observed at high potential and a distinctive peak increasing over time was recorded on cyclic voltamogramms, suggesting the growth of oxygen reducing microbes. 16S pyrotag sequencing showed the enrichment of two operational taxonomic units (OTUs) affiliated to Ectothiorodospiraceae on high potential electrodes with the best performances. Shotgun metagenome sequencing and a newly developed method for the identification of Taxon Specific Gene Annotations (TSGA) revealed Ectothiorhodospiraceae specific genes possibly involved in electron transfer and in autotrophic growth. These results give interesting insights into the genetic features underlying the selection of efficient oxygen reducing microbes on biocathodes

Whole proteome analyses on Ruminiclostridium cellulolyticum show a modulation of the cellulolysis machinery in response to cellulosic materials with subtle differences in chemical and structural properties

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the xyl-doc cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.This work was supported by a grant [R2DS 2010-08] from Conseil Regional d'Ile-de-France through DIM R2DS programs (http://www.r2ds-ile-de-france.com/). Irstea (www.irstea.fr/) contributed to the funding of a PhD grant for the first author. The funders provided support in the form of salaries for author [NB], funding for consumables and laboratory equipment, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Omics Services provided support in the form of salaries for authors [VS, MD], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors [NB, VS, MD] are articulated in the 'author contributions' section.info:eu-repo/semantics/publishedVersio

Groupes microbiens fonctionnels impliqués dans la méthanisation de la cellulose et du méthanol (diversité, fonction et influence de la température)

Afin de mieux comprendre le fonctionnement des communautés microbiennes intervenant lors de la méthanisation de la cellulose et du méthanol, des échantillons issus de la digestion anaérobie de déchets ont été incubés en présence de cellulose, de glucose, d'acétate et de méthanol enrichis en carbone 13, en conditions mésophiles et thermophiles. Après extraction et séparation de l'ADN lourd et léger par ultracentrifugation sur des gradients de densité (technique SIP), la diversité fonctionnelle a été caractérisée par inventaire moléculaire et analyse phylogénétique. Au total, 20 banques de clones contenant des gènes d'ADNr 16S bactériens (1251 séquences) et 19 banques de clones contenant des gènes ADNr 16S archéens (1312 séquences) ont été obtenues en conditions mésophiles et thermophiles. Des oligonucléotides spécifiques ont ensuite été élaborés et hybridés selon la technique d'hybridation fluorescente in situ afin de confirmer la présence des groupes fonctionnels pré-identifiés. Nous nous sommes ensuite attachés à étudier la relation entre la cinétique de dégradation, les voies métaboliques activées et les groupes fonctionnels impliqués, en suivant la dynamique des diversités fonctionnelles à l'aide de la technique du polymorphisme de conformation simple brin (SSCP). En appliquant de manière combinée ces techniques sur une série de substrats fonctionnellement connectés, nous dévoilons des réseaux de microorganismes non cultivables impliqués dans la méthanisation de la cellulose et du méthanol. La température d'incubation influence la diversité générale tandis que l'ajout de substrats joue un rôle important sur la diversité fonctionnelle. Les microorganismes appartenant au genre Acetivibrio et à l'ordre Halanaerobiales sont, respectivement, les principaux groupes hydrolysant la cellulose en conditions mésophiles et thermophiles. Les microorganismes appartenant à la famille Porphyromonadaceae et au genre Clostridium sont les principaux fermenteurs du glucose. Par ailleurs, de nombreux microorganismes appartenant à la classe Clostridia, mais éloignés de toutes les souches cultivables répertoriées, ont été identifiés comme des groupes fonctionnels dans toutes les incubations. L'oxydation syntrophique de l'acétate est un processus important dans cet écosystème, qui pourrait être effectué par des microorganismes proches du genre Pseudomonas en conditions mésophiles. Une grande versatilité fonctionnelle a été mise en évidence pour les microorganismes thermophiles et pourrait contribuer à expliquer une dégradation de la cellulose plus rapide et efficace par rapport à celle observée en conditions mésophiles. Enfin, une nouvelle technique, baptisée "SIMSISH" (Secondary Ion Mass Spectrometry In Situ Hybridization), a été développée dans le cadre du travail. Elle permet de visualiser simultanément l'identité microbienne et de mesurer l'enrichissement isotopique à l'échelle d'une cellule. Couplée aux méthodologies SIP, cette technique devrait permettre des progrès décisifs pour l'étude in situ de la contribution des populations microbiennes non cultivables au fonctionnement des écosystèmes complexes.In order to better understand functional communities of microbes catalyzing methanization of cellulose and methanol, samples from municipal solid waste anaerobic digester were incubated with 13C-cellulose, 13C-glucose, 13C-acetate and 13C-methanol under mesophilic and thermophilic conditions. Firstly, the functional diversity was characterized by coupling DNA stable isotope probing (SIP) to phylogenetic analysis. Totally 20 bacterial clone libraries (1251 sequences) and 19 archaeal clone libraries (1312 sequences) of 16S rRNA gene were obtained for both mesophilic and thermophilic conditions. Then, specific FISH probes were designed and hybridized to confirm the presence in situ of pre-identified functional groups. Secondly, we studied the relationships between degradation kinetics, metabolic pathways and microbial functional diversity dynamics, using time-course DNA-SIP coupled to PCR-single strand conformation polymorphism. Thanks to the combination of these techniques on a series of functionally connected substrates, we shed light on the networks of uncultured microbes catalyzing the methanization of cellulose and methanol. Incubation temperature shaped the general microbial diversity while the added substrates played an important role on the functional diversity. Microorganisms affiliated with the genus Acetivibrio and the order Halanaerobiales were respectively the principal cellulose hydrolyzers under mesophilic and thermophilic conditions, whereas microorganisms affiliated with the family Porphyromonadaceae and the genus Clostridium were the main glucose fermenters. Besides, large amount of microorganisms affiliated with class Clostridia, but distantly related to any hitherto cultured strain, were identified as functional groups in all degradation experiments. Syntrophic oxidation of acetate was an important process in this ecosystem both under mesophilic and thermophilic conditions. Interestingly, our results suggest that Pseudomonas-related microorganisms were involved in this process under mesophilic conditions. A high functional versatility of microbial groups was evidenced under thermophilic conditions which could contribute to a more rapid and efficient cellulose degradation compared to mesophilic conditions. Finally, a new technique called SIMSISH was developed during this work providing the possibility to simultaneously visualize the microbial identity and measure the isotopic enrichment at the single cell level. Combined to SIP analysis, this technique might allow a decisive step forward for the study of the function of uncultured microbes within their environments.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Fractionnements isotopiques (13C/12C) engendrés par la méthanogenèse (apports pour la compréhension des processus de biodégradation lors de la digestion anaérobie)

Les procédés anaérobies de traitement de déchets apparaissent clairement pouvoir répondre à l'enjeu socio-économique actuel que représente la valorisation énergétique de la fraction organique contenue dans les déchets ménagers. En effet, les processus de dégradation anaérobies font intervenir en cascade, différentes réactions et populations de micro-organismes permettant de transformer la matière organique en biogaz riche en méthane. Une bonne connaissance des effets des paramètres opérationnels sur l'orientation des métabolismes s'avère ainsi nécessaire à l'émergence de solutions permettant d'optimiser ces procédés. Ceci est notamment le cas pour la dernière étape, appelée méthanogenèse. Dans ce contexte, l'approche isotopique reposant sur la mesure de la composition isotopique (13C/12C) du méthane et du dioxyde de carbone, devrait pouvoir répondre à cet objectif en permettant l'identification des métabolismes à l'origine de la production du méthane. La transposabilité à l'étude de la digestion anaérobie des déchets de cette approche isotopique déjà utilisée dans les écosystèmes naturels, a tout d'abord été vérifiée expérimentalement. Les effets de certains paramètres opérationnels connus pour avoir un impact fort sur le processus de digestion anaérobie, tels que la température et la concentration en azote ammoniacal, ont ensuite été étudiés. Il a été mis en évidence qu'en condition thermophile, la méthanogenèse acétoclaste observée en condition mésophile, était remplacée par une oxydation syntrophique de l'acétate lors de la digestion anaérobie des déchets ménagers. Des expériences sur acétate ont montré que cet effet sur les voies métaboliques n'était toutefois pas systématique et pourrait ne pas être dû à un effet direct d'une augmentation de la température, mais plutôt à l'accroissement de la concentration en ammoniaque qui en résulte. D'autres expériences ont clairement établi qu'une augmentation de la concentration en azote ammoniacal conduisait également à la mise en place de l'oxydation syntrophique de l'acétate. Le couplage de l'approche isotopique avec des analyses microbiologiques a révélé que cette réaction d'oxydation syntrophique de l'acétate, à haute concentration en azote ammoniacal, pouvait s'établir telle que déjà décrite, par la mise en place d'une relation symbiotique bactéries/archées hydrogénotrophes strictes, mais également de manière différente en impliquant des membres de la famille Methanosarcinaceae qui pourraient réaliser seuls les deux étapes de la réaction (oxydation et méthanogenèse hydrogénotrophe). L'application de l'approche isotopique a également permis de mettre en évidence, lors d'une expérience visant à simuler la recirculation de différents effluents au sein d'une installation de stockage de déchets bioactive, l'influence de la nature de l'effluent sur l'orientation des métabolismes méthanogènes. Enfin, l'influence de la proportion de déchets verts, lors de la co-digestion biodéchets / déchets verts, sur la concentration en ions ammonium libérés ainsi que sur l'orientation du métabolisme en résultant, a été étudiée. Les potentialités d'une utilisation de l'approche isotopique sur site ont également été investiguées au travers d'une campagne de mesures sur une installation de stockage de déchets non dangereux.Anaerobic waste treatment processes are clearly part of the answer to a current important socio-economic issue in waste management: energy production from the organic fraction of municipal solid waste. The anaerobic digestion of municipal solid waste is a complex process involving numerous reactions and microorganism communities. At the end of the degradation process, some biogas with a particularly high methane content is produced. A detailed knowledge on how operational parameters affect metabolism orientations is required to optimize these treatment processes. This is in particular the case for the last degradation reaction called methanogenesis. In this context, an isotopic approach based on isotopic composition measurements (13C/12C) for methane and carbon dioxide can provide some clues with regard to this objective. Indeed, this methodology enables the determination of the methanogenic pathways by which methane is produced.Transferability of the isotopic approach used for natural ecosystems to the field of anaerobic digestion of municipal solid waste was first experimentally verified. In a second time, the effects of some operational parameters known to strongly impact the anaerobic digestion process, such as temperature and ammonia concentration, were studied. During anaerobic digestion of reconstituted municipal solid waste in thermophilic conditions, it was shown that aceticlastic methanogenesis (occurring in mesophilic conditions) was replaced by a syntrophic acetate oxidation reaction. Additional experiments using acetate as sole substrate were performed and showed that this effect on the metabolic pathways was not systematic. Consequently, it cannot be due to a direct effect of the temperature increase. It could rather be explained by the induced and indirect increase in ammonia concentration. Additional experiments clearly demonstrated that an increase in ammonia concentration led to the establishment of a syntrophic acetate oxidation reaction. The isotopic approach was combined with microbiological analyses and showed that the syntrophic acetate oxidation reaction occurring at high ammonia concentration during acetate incubations could have been performed through a syntrophic relationship between bacteria and strict hydrogenotrophic archaea, as previously described in the literature. Interestingly, the syntrophic acetate oxidation could also have occurred using a different pathway relying on members of the Methanosarcinaceae family putatively able to perform the two steps of the reaction (oxidation and hydrogenotrophic methanogenesis). In addition, the implementation of the isotopic approach during an experiment designed to simulate a landfill bioreactor evidenced the influence of the effluent's nature on the methanogenesis metabolism orientation. The influence of green waste proportion during the co-digestion of biowaste / green waste mixtures on resulting ammonia concentrations and methanogenesis pathways was also studied through dedicated experiments. Finally, the potential of the isotopic approach for landfill-scale application was investigated through a measurement campaign on a landfill site.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

A model-based approach to detect interspecific interactions during biofilm development

A model-based approach was developed to detect interspecific interactions during biofilm development. This approach relied on the comparison of experimental data with a simple null model of biofilm growth dynamics where individual species grew independently of one another, except that they competed for space. Such a model was directly parameterized with a 4D confocal image series of biofilms and then used as a null model to detect interspecific interactions between pairs of bacterial species. This approach was tested in two bispecific competitive trials. In the first trial, the progressive exclusion of Pseudomonas fluorescens by Pseudomonas putida appeared to be due solely to the different intrinsic growth rates of the two strains. In contrast, modelling results suggested the presence of interference competition between Pseudomonas aeruginosa and P. putida in mixed biofilms. The authors' approach enables the detection of ecologically relevant interactions which constitute a prerequisite to building a comprehensive view of the dynamics and functioning of spatially structured bacterial communities

Comparison of conventional and new energy-based models for modeling activated sludge dynamics

International audienc

Insights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions.

International audienceDNA-SIP (stable isotope probing) was conducted on anaerobic municipal solid waste samples incubated with (13)C-cellulose, (13)C-glucose and (13)C-acetate under mesophilic conditions. A total of 567 full-length bacterial and 448 1100-bp-length archaeal 16S rRNA gene sequences were analysed. In the clone libraries derived from 'heavy' DNA fractions, the most abundant sequences were affiliated with the phyla Firmicutes, Bacteroidetes, the gamma-subclass of Proteobacteria and methanogenic orders Methanomicrobiales and Methanosarcinales. Sequences related to the genus Acetivibrio (phylum Firmicutes) were recovered only in the 'heavy' DNA fraction derived from the (13)C-cellulose incubation. An oligonucleotide probe (UCL284) targeting specifically Acetivibrio was designed and used for fluorescent in situ hybridization (FISH) experiments. Interestingly, hybridization of the probe was detected in microorganisms aggregated around cellulose fibres, strengthening the conclusion that these microorganisms were major cellulose degraders. Sequences related to genus Clostridium (phylum Firmicutes) and to the family Porphyromonadaceae (phylum Bacteroidetes) were retrieved in large numbers from the 'heavy' DNA library of (13)C-Glucose incubation, suggesting their involvement in saccharide fermentation. Design and hybridization of specific FISH-probes confirmed the abundant representation of Clostridium (CLO401, CLO1248) and Porphyromonadaceae (BAC1040), which were mostly observed in the planktonic phase. Surprisingly, in the (13)C-acetate experiment, the 'heavy' DNA archaeal library was dominated by sequences related to the strictly hydrogenotrophic methanogenic genus Methanoculleus. One single operational taxonomic unit containing 70 sequences, affiliated to the gamma-subclass of Proteobacteria, was retrieved in the corresponding bacterial library. FISH observations with a newly designed specific probe (UGA64) confirmed the dominance of this bacterial group. Our results show that combination of DNA-SIP and FISH applied with a series of functionally connected substrates can shed light on the networks of uncultured microbes catalysing the methanization of the most abundant chemical renewable energy source on Earth