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

The stromal side of the cytochrome b6f complex regulates state transitions

International audienceIn oxygenic photosynthesis, state transitions distribute light energy between PSI and PSII. This regulation involves reduction of the plastoquinone pool, activation of the state transitions 7 (STT7) protein kinase by the cytochrome (cyt) b6f complex, and phosphorylation and migration of light harvesting complexes II (LHCII). In this study, we show that in Chlamydomonas reinhardtii, the C-terminus of the cyt b6 subunit PetB acts on phosphorylation of STT7 and state transitions. We used site-directed mutagenesis of the chloroplast petB gene to truncate (remove L215b6) or elongate (add G216b6) the cyt b6 subunit. Modified complexes are devoid of heme ci and degraded by FTSH protease, revealing that salt bridge formation between cyt b6 (PetB) and Subunit IV (PetD) is essential to the assembly of the complex. In double mutants where FTSH is inactivated, modified cyt b6f accumulated but the phosphorylation cascade was blocked. We also replaced the arginine interacting with heme ci propionate (R207Kb6). In this modified complex, heme ci is present but the kinetics of phosphorylation are slower. We show that highly phosphorylated forms of STT7 accumulated transiently after reduction of the PQ pool and represent the active forms of the protein kinase. The phosphorylation of the LHCII targets is favored at the expense of the protein kinase, and the migration of LHCII toward PSI is the limiting step for state transitions

Ecological consequences of abrupt temperature changes in anaerobic digesters

International audienceTemperature is a key parameter of anaerobic digestion. Its modification can have drastic consequences on microbial communities and performances. However it can be an interesting parameter to adjust the productivity, energy efficiency or stability of bioreactors. The objective of this work was to give insights into the consequences of abrupt temperature modifications on the microbiota of anaerobic digestion. Two complementary experiments were performed. A continuously stirred lab-scale bioreactor (5-L) simulated the functioning of a semi-continuous industrial digester. Five batch-fed incubations (1-L) served as replicated experiments. Each experiment was divided into three successive steps: reactors were first operated at 35 degrees C followed by an abrupt increase to 55 degrees C and finally a decrease to 35 degrees C. After the first temperature shock, gas production rate increased temporarily and then fell for 1 month before a new steady-state was reached. When temperature was reset to 35 degrees C in step 3 gas production decreased sharply again. The semi-continuous reactor recovered after 15 days while the batch-fed incubations never recovered. Ecological changes associated to these performance drops and recovery were sought using 16S sequencing of Bacteria and Archaea and multivariate analyses. In brief, Bacteroidales order was rapidly and strongly affected by temperature increase, while Clostridiales became dominant in thermophilic conditions. Several thermotolerant Bacteria were identified as responsible for reactors early recovery, but were outcompeted by a very diverse bacterial population a few weeks after temperature shock. Methanosarcina and Methanobacterium Archaea dominated at 35 degrees C but were slowly replaced by thermophilic Methanoculleus or Methanosarcina at 55 degrees C that were essential to methane production. In step 3, when returning to initial temperature conditions after the thermophilic period, Ruminococcaceae and Methanobacterium appeared to drive digester resilience

Integrative Analyses to Investigate the Link between Microbial Activity and Metabolite Degradation during Anaerobic Digestion

International audienceAnaerobic digestion (AD) is a promising biological process that converts waste into sustainable energy. To fully exploit AD's capability, we need to deepen our knowledge of the microbiota involved in this complex bioprocess. High-throughput methodologies open new perspectives to investigate the AD process at the molecular level, supported by recent data integration methodologies to extract relevant information. In this study, we investigated the link between microbial activity and substrate degradation in a lab-scale anaerobic codigestion experiment, where digesters were fed with nine different mixtures of three cosubstrates (fish waste, sewage sludge, and grass). Samples were profiled using 16S rRNA sequencing and untargeted metabolomics. In this article, we propose a suite of multivariate tools to statistically integrate these data and identify coordinated patterns between groups of microbial and metabolic profiles specific of each cosubstrate. Five main groups of features were successfully evidenced, including cadaverine degradation found to be associated with the activity of microorganisms from the order Clostridiales and the genus Methanosarcina. This study highlights the potential of data integration toward a comprehensive understanding of AD microbiota

Colonization kinetics and implantation follow-up of the sewage microbiome in an urban wastewater treatment plant

The Seine-Morée wastewater treatment plant (SM_WWTP), with a capacity of 100,000 population-equivalents, was fed with raw domestic wastewater during all of its start-up phase. Its microbiome resulted from the spontaneous evolution of wastewater-borne microorganisms. This rare opportunity allowed us to analyze the sequential microbiota colonization and implantation follow up during the start-up phase of this WWTP by means of regular sampling carried out over 8 months until the establishment of a stable and functional ecosystem. During the study, biological nitrification-denitrification and dephosphatation occurred 68 days after the start-up of the WWTP, followed by flocs decantation 91 days later. High throughput sequencing of 18S and 16S rRNA genes was performed using Illumina's MiSeq and PGM Ion Torrent platforms respectively, generating 584,647 16S and 521,031 18S high-quality sequence rDNA reads. Analyses of 16S and 18S rDNA datasets show three colonization phases occurring concomitantly with nitrification, dephosphatation and floc development processes. Thus, we could define three microbiota profiles that sequentially colonized the SM_WWTP: the early colonizers, the late colonizers and the continuous spectrum population. Shannon and inverse Simpson diversity indices indicate that the highest microbiota diversity was reached at days 133 and 82 for prokaryotes and eukaryotes respectively; after that, the structure and complexity of the wastewater microbiome reached its functional stability. This study demonstrates that physicochemical parameters and microbial metabolic interactions are the main forces shaping microbial community structure, gradually building up and maintaining a functionally stable microbial ecosystem

Shotgun metaproteomic profiling of biomimetic anaerobic digestion processes treating sewage sludge

Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two continuous stirred tank reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After 3 months of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein-inferred and 16S ribosomal DNA tags based taxonomic community profiles were not consistent. PCA however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow-derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4 × 10(6) reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (11 vs. 4% of spectral identification rate for the combined samples). Surprisingly, a high proportion of proteins from members of the "Candidatus Competibacter" group, a key microbial player usually found in activated sludge plants, was retrieved in our anaerobic digester samples. Data are available via ProteomeXchange with identifier PXD00242

A longitudinal study of the effect of temperature modification in full-scale anaerobic digesters – dataset combining 16S rDNA gene sequencing, metagenomics, and metabolomics data

International audienceData in this article provides detailed information on the microbial dynamics and degradation performances in two fullscale anaerobic digesters operated in parallel for 476 days. One of them was kept at 35 °C for the whole experiment, while the other was submitted to sub-mesophilic (25 °C

Metataxonomics, metagenomics, metabolomics analysis of the influence of temperature modification in full-scale anaerobic digesters

International audienceFull-scale anaerobic digesters' performance is regulated by modifying their operational conditions, but little is known about how these modifications affect their microbiome. In this work, we monitored two originally mesophilic (35°C) full-scale anaerobic digesters during 476 days. One digester was submitted to sub-mesophilic (25°C) conditions between days 123 and 373. We characterized the effect of temperature modification using a multi-omics (metataxonomics, metagenomics, and metabolomics) approach. The metataxonomics and metagenomics results revealed that the lower temperature allowed a substantial increase of the sub-dominant bacterial population, destabilizing the microbial community equilibrium and reducing the biogas production. After restoring the initial mesophilic temperature, the bacterial community manifested resilience in terms of microbial structure and functional activity. The metabolomic signature of the sub-mesophilic acclimation was characterized by a rise of amino acids and short peptides, suggesting a protein degradation activity not directed towards biogas production

Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers.

International audienceEnhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes

Combination of bioanode and biocathode for the conversion of wastes into biocommodities using microbial electrosynthesis

PosterBioelectrochemical systems (BES) as microbial fuel cells take advantages of microorganisms to convert the chemical energy of organic waste into electricity. Recently, the discovery that BES can also be used for the synthesis of biocommoditiesvia microbial electrosynthesis (MES) has greatly expanded the horizons for their applications. Indeed, some microbes are able to use electrons and molecules such as CO2 to synthesize reduced products: volatile fatty acids, alcohols etc... By combining both these processes, it should thus theoretically be possible to use the electrons of organic waste to synthesize bio-based chemicals in a clean and controlled compartment. However, these technologies are only few years old and required scientific data before they can be practically applied. In this context, we developed a dual-chamber reactor with both biotic anode (carbon cloth) and cathode (stainless steel) separated by a cation-exchange membrane. Bioanode was inoculated using an anodic biofilm sample formed in biological wastes and biocathode by injecting a suspension of a homoacetogen-enriched culture. Acetate (600mg/l) was used as electron donor in the anodic compartment. Chronoamperometry experiments were carried out with a multi-channel potentiostat in order to monitor electroactivity of the microbial communities. Anode potential was poised at +0.158 volts versus saturated calomel electrode (SCE) for startup. Chemical analyses (volatile fatty acids (VFAs), cations/anions, chemical oxygen demand, and total organic carbon) were performed to evaluate the metabolic pathways. Microbial community diversity was investigated by 16S rDNA pyrosequencing (MiSeq sequencer, Illumina®). After the total consumption of acetate at anode (run 1), a second run (run 2) was launched by re-injecting 600 mg/L of acetate in anodic compartment and also 2-bromo-ethane sulfonate (2-BES) at the cathode to inhibit methanogenesis. Current density of 5 A/m² was reached after 24h of experiment. During run 1 and 2 78% and 89% of electrons from acetate were respectively transferred in the system revealing satisfying coulombic efficiency at the bioanode. During run 1, incoming electrons at biocathode were mainly used to produce methane (53% of total incoming electrons) and only traces of VFAs were detected. However, at the end of the second run, VFAs accumulated with a production rate of acetate reaching 11 g.m².d-1 and corresponding to 29% of the electrons coming from the anode. Using gas chromatography coupled with mass spectrometry, caprylate (C8H16O2) was also detected in reactors showing the ability of the MES system to produce molecules with an elongated carbon chain. Microbial diversity profiles showed a switch of archaeal and bacterial populations in cathodic compartment between run 1 and run 2 suggesting that VFAs production resulted from microbial adaptation to the addition of BES in the cathodic compartment. Overall this work constitutes a first step toward the utilization of MES systems for the conversion of organic wastes into biofuels and chemicals using coupled bioanode and biocathode