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Metabolically active microorganisms are increasingly acknowledged as actors of cloud chemical reactivity able to use organic compounds present in clouds (e.g. organic acids, aldhedydes) for their metabolism (Vaïtilingom et al., 2013). Uncharacterized biological activity may play a major role especially during the night, while during daytime the abiotic degradation of organic compounds would be driven and dominated by hydroxyl radical (•OH) chemistry (Vaïtilingom et al., 2011). To better understand and predict the impact of biological activity on atmospheric chemical reactivity, the metabolic pathways of the whole cloud microbiome and their modulations by environmental conditions (temperature, light, oxidants) must now be assessed.The METACLOUD project addresses metabolic acclimatation of cloud microorganisms under two contrasted situations simulating a summer day (17°C, with solar light and presence of hydrogen peroxide) and a winter night (at 5°C, in the dark and without hydrogen peroxide). A focus is made on formaldehyde assimilations as this compound is a key intermediate both in cloud radical chemistry and in many C1 biological pathway, using fluxomics (LC-HRMS and IC-HRMS) on 13C-formaldehyde supplemented samples. Experiments were conducted in specially designed photobioreactors, either on (1) freshly sampled cloud water from the research station at the top of the puy de Dôme station (1465m asl, PUY, France) including naturally present microorganisms, or (2) an artificial consortium assembled from microbial strains isolated from cloud water sampled at PUY and resuspended in an artificial medium mimicking the composition of marine cloud water (major inorganic and organic compounds).Metatranscriptomes and metabolomes indicate metabolic acclimations of the cloud microbiome to model summer/winter conditions, especially linked with fatty acid regulation and central metabolism (e.g. citrate cycle). First results with 13C-formaldehyde showed carbon incorporation from this molecule into several classes of metabolites (e.g. nucleotides, amino acids, central metabolites), illustrating the complex biological fate of this compound in the environment. The data will be used to implement biological activity on cloud chemistry models. Vaïtilingom M. et al. (2011) Atmospheric chemistry of carboxylic acids: microbial implication versus photochemistry. Atmos. Chem. Phys. 11, 8721-8733. doi: 10.5194/acp-11-8721-2011.Vaïtilingom M. et al. (2013) Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds. Proc. Nat. Acad. Sci. USA 110, 559-564. doi: 10.1073/pnas.1205743110.

Microbiology still relies on en masse cultivation for selection, isolation, and characterization of microorganisms of interest. This constrains the diversity of microbial types and metabolisms that can be investigated in the laboratory also because of intercellular competition during cultivation. Cell individualization by droplet-based microfluidics prior to experimental analysis provides an attractive alternative to access a larger fraction of the microbial biosphere, miniaturizing the required equipment and minimizing reagent use for increased and more efficient analytical throughput. Here, we show that cultivation of a model two-strain bacterial community in droplets significantly reduces representation bias in the grown culture compared to batch cultivation. Further, and based on the droplet shrinkage observed upon cell proliferation, we provide proof-of-concept for a simple strategy that allows absolute quantification of microbial cells in a sample as well as selective recovery of microorganisms of interest for subsequent experimental characterization.

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de Dôme (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56 Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

The complete genome of Sphingomonas aerolata PDD-32b-11, a bacterium isolated from cloud water, was sequenced. It features four circular replicons, a chromosome of 3.99 Mbp, and three plasmids. Two putative rhodopsin-encoding genes were detected which might act as proton pumps to harvest light energy.

International audience; Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation suggests a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as a sole source of carbon and energy for growth, Methylorubrum extorquens DM4 is a longstanding reference strain. Here, the primary 5′-ends of transcripts were obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM or methanol. In total, 7231 putative TSSs were annotated and classified with respect to their localization to coding sequences (CDSs). TSSs on the opposite strand of CDS (antisense TSS) account for 31% of all identified TSSs. One-third of the detected TSSs were located at a distance to the start codon inferior to 250 nt (average of 84 nt) with 7% of leaderless mRNA. Taken together, the global TSS map for bacterial growth using DCM or methanol will facilitate future studies in which transcriptional regulation is crucial, and efficient DCM removal at polluted sites is limited by regulatory processes.

International audience; The soil dissipation of the widely used herbicides S-metolachlor (SM) and butachlor (BUT) was evaluated in laboratory microcosms at two environmentally relevant doses (15 and 150 mg/g) and for two agricultural soils (crop and paddy). Over 80% of SM and BUT were dissipated within 60 and 30 days, respectively, except in experiments with crop soil at 150 mg/g. Based on compound-specific isotope analysis (CSIA) and observed dissipation, biodegradation was the main process responsible for the observed decrease of SM and BUT in the paddy soil. For SM, biodegradation dominated over other dissipation processes, with changes of carbon isotope ratios (Dd 13 C) of up to 6.5‰ after 60 days, and concomitant production of ethane sulfonic acid (ESA) and oxanilic acid (OXA) transformation products. In crop soil experiments, biodegradation of SM occurred to a lesser extent than in paddy soil, and sorption was the main driver of apparent BUT dissipation. Sequencing of the 16S rRNA gene showed that soil type and duration of herbicide exposure were the main determinants of bacterial community variation. In contrast, herbicide identity and spiking dose had no significant effect. In paddy soil experiments, a high (4:1, V/V) ESA to OXA ratio for SM was observed, and phylotypes assigned to anaerobic Clostridiales and sulfur re-ducers such as Desulfuromonadales and Syntrophobacterales were dominant for both herbicides. Crop soil microcosms, in contrast, were associated with a reverse, low (1:3, V/V) ratio of ESA to OXA for SM, and Alphaproteobacteria, Actinobacteria, and Bacillales dominated regardless of the herbicide. Our results emphasize the variability in the extent and modes of SM and BUT dissipation in agricultural soils, and in associated changes in bacterial communities.

Chloromethane (CH3Cl) is an important source of chlorine in the stratosphere, but detailed knowledge of the magnitude of its sources and sinks is missing. Here, we measured the stable chlorine isot...

Dichloromethane (DCM) is a toxic industrial solvent frequently detected in multi-contaminated aquifers. It can be degraded biotically or abiotically, and under oxic or anoxic conditions. The extent and pathways of DCM degradation in aquifers may thus depend on water table fluctuations and microbial responses to hydrochemical variations. Here, we examined the effect of water table fluctuations on DCM biodegradation in two laboratory aquifers fed with O2-depleted DCM-spiked groundwater from a well-characterized former industrial site. Hydrochemistry, stable isotopes of DCM (δ13C and δ37Cl), and bacterial community composition were examined to determine DCM mass removal and degradation pathways under steady-state (static water table) and transient (fluctuating water table) conditions. DCM mass removal was more pronounced under transient (95%) than under steady-state conditions (42%). C and Cl isotopic fractionation values were larger under steady-state ( e bulk C = −23.6 ± 3.2‰, and e bulk Cl = −8.7 ± 1.6‰) than under transient conditions ( e bulk C = −11.8 ± 2.0‰, and e bulk Cl = −3.1 ± 0.6‰). Dual C-Cl isotope analysis suggested the prevalence of distinct anaerobic DCM degradation pathways, with ΛC/Cl values of 1.92 ± 0.30 and 3.58 ± 0.42 under steady-state and transient conditions, respectively. Water table fluctuations caused changes in redox conditions and oxygen levels, resulting in a higher relative abundance of Desulfosporosinus (Peptococcaceae family). Taken together, our results show that water table fluctuations enhanced DCM biodegradation, and correlated with bacterial taxa associated with anaerobic DCM degradation. Our integrative approach allows to evaluate anaerobic DCM degradation under dynamic hydrogeological conditions, and may help improving bioremediation strategies at DCM contaminated sites.

International audience; A liquid/gas membrane bioreactor (L/G MBR) was developed to intensify the dark fermentation process. A hollow fiber membrane module was used to combine biohydrogen production, in situ liquid–gas separation and hydrogen producing bacteria retention in a single unit. The L/G MBR was seeded once and did not require further microbial input, as consistent average hydrogen yield of 0.97 ± 0.09 mol-H2/added mol-glucose and hydrogen production rate of 106.5 ± 10.6 mL-H2/L-medium/h were reached over a year. Different biogas extraction strategies showed that efficient in situ H2 extraction is possible without sweeping gas in the lumen of the fibers, thus facilitating H2 purification in an industrial setting. Modelling predicted an optimal hydrogen yield of 1.2 mol/mol-glucose added for a glucose concentration in the feed of 13.1 g/L, close to experimental hydraulic retention time of 8–10 h with an organic loading rate of 1.4 g-glucose/L-medium/h. No washout of hydrogen-producing bacteria was observed at low HRT (2 h), suggesting the possibility of further hydrogen production rate enhancement using an optimized organic loading rate. Acetate and butyrate were the main metabolites identified. Clostridium and Enterobacter dominated in the liquid outlet. The relative abundance of Clostridium pasteurianum increased with glucose concentration in the bioreactor, as opposed to Clostridium beijerinckii which was more abundant at low glucose concentration. The original hollow fiber L/G MBR configuration enabled the testing and selection of fermentation strategies that greatly simplified the implementation of the dark fermentation process by addressing its key operational bottlenecks. Indeed, the L/G membrane surface served as a support and reservoir for the hydrogen producing bacteria across a wide range of HRT conditions.

A Cr6+ resistant Microbacterium testaceum B-HS2, isolated indigenously from tannery wastewater, showed optimum growth at 37 °C and pH 7. M. testaceum B-HS2 could resist to Cr6+ (48 mM) and heavy metals upto 2 mM (As2+, Zn2+, Cu2+), 7 mM (Pb2+) and 1 mM (Cd2+, Ni2+). Maximum activity of chromate reductase was achieved at 40 °C at pH of 7 and was inhibited in presence of all the heavy metals tested. M. testaceum B-HS2 biosorption efficiency (q) for Cr6+ was 31, 38, 66 and 47 mM/g after 2, 4, 6 and 8 days, respectively. Electron micrographs confirmed further the adsorption of metal leading to intracellular accumulation of Cr6+. Functional groups such as amide and carbonyl moieties which actively participated in Cr6+ adsorption were determined through FTIR spectroscopy, and intracellular accumulation was also confirmed by energy dispersive X-ray (EDX) and scanning electron microscopy (SEM) analysis. Cr6+ presence triggers significant production of antioxidant enzymes [(Ascorbate peroxidase (APOX), Superoxide dismutase (SOD), Peroxidase (POX), Glutathione S-transferease (GST), and Catalase (CAT)]. Moreover, rise in glutathione and other non-protein thiol levels were determined which substantially neutralize Cr6+ generated oxidative stress. Pilot scale study revealed that M. testaceum B-HS2 was helpful in removing up to 96% Cr6+ from tannery effluent within 6 days and this microbial purified water is safe for the plant growth. Multiple heavy metal tolerance and high Cr6+ reduction potential make M. testaceum B-HS2 a candidate of choice to reclaim Cr6+ contaminated environment.

The genus Paenibacillus was hardly described as a Fe(III)-reducing agent, only limited to reduce soluble forms or Fe inserted in poorly crystallized structures. In this study, three Paenibacillus strains capable of reducing manganese oxides in addition to iron oxides were isolated from Cameroonian and Brazilian soils. These strains reduced iron minerals from poorly crystallized 2-line ferrihydrite to well-crystallized Al-substituted and pure goethite with a significant production of soluble ferrous iron. These Paenibacillus strains, inhabitants from ferralitic temporarily waterlogged soils, could play an important role in the bioweathering of minerals and metal mobility in soils.

Background: Chloromethane (CH3Cl) is the most abundant chlorinated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. Results and Conclusions: We investigated chloromethane-degrading taxa associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13C-labelled CH3Cl combined with metagenomic DNA sequencing. Metagenome assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterised methylotrophic cmuA-independent pathway likely drives CH3Cl degradation in the investigated tree ferns.

International audience; The aim of this work was to assess the performances of wine byproduct biomass for hydrogen production by dark fermentation. Grape must deposits from two grape varieties (Pinot Gris and Chardonnay) were considered, either with external microbial inoculum or without. We show that grape must residues contain endogenous microflora, well adapted to their environment, which can degrade sugars (initially contained in the biomass) to hydrogen without any nutrient addition. Indeed, hydrogen production during endogenous fermentation is as efficient as with an external heat-treated inoculum (2.5 ± 0.4 LH2.L-1reactor and 1.61 ± 0.41 molH2.mol-1consumed hexose, respectively) with a lower energy cost. Hydrogen-producing bacteria were selected from the endogenous microflora during semi-batch bioreactor operation, as shown by T-RFLP profiles and 16S rRNA sequencing, with Clostridium spp. (butyricum, beijerinckii, diolis, roseum) identified as the major phylotype. Such hydrogen production efficiency opens new perspectives for innovating in the valorization of winery by-products.

International audience; Chloromethane (CH3 Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3 Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3 Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3 Cl-degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13 C-labelled CH3 Cl combined with metagenomics. Metagenome-assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3 Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3 Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3 Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA-independent pathway may drive CH3 Cl degradation in the investigated tree ferns.

Dichloromethane (DCM) is a toxic industrial solvent frequently detected in multi-contaminated aquifers. DCM often co-occurs with chlorinated ethenes resulting in complex mixtures posing challenges to predict its fate in groundwater. Changes in hydrochemistry and redox conditions in groundwater due to fluctuations in the water table may affect the extent and pathways of pollutant biodegradation. In this context, Compound-Specific Isotope Analysis (CSIA) is a useful tool to evaluate natural degradation of halogenated hydrocarbons. In this study, the impact of water table fluctuations on DCM biodegradation was examined in two laboratory aquifers using dual-element isotope analysis - the stable isotope fractionation of two elements (e.g., 13C and 37Cl), and high-throughput biomolecular approaches. The aquifers were supplied with contaminated groundwater from the former industrial site Thermeroil (France). High-resolution sampling and monitoring of pore water allowed examining, under steady and transient conditions, the aquifers response with respect to hydrochemistry and microbial composition. A dual C-Cl stable isotope approach (ΛC/Cl = Δδ13C/Δδ37Cl) was developed using GC-IRMS (C-DCM) and GC-MS (Cl-DCM) to estimate the extent of DCM degradation and to identify DCM degradation pathways. Under the experimental steady conditions, dissolved oxygen (2+ concentrations at lower depths of the aquifer models indicated iron-reducing prevailing conditions, while mass transfer of oxygen increased during water table fluctuations. Pronounced carbon isotope fractionation of DCM was associated with larger DCM mass removal under transient conditions (>90%) compared to steady conditions (mass removal of 35%). Under transient conditions, carbon enrichment factors (εC) became larger over time ranging from -18.9 ± 3.4‰ to -33 ± 0.3‰ whereas chlorine enrichment factors (εCl) remained constant (-3.6 ± 0.7‰). In contrast, a similar εC of -20 ± 3.5‰ (beginning of transient condition) but a larger εCl of -10.8 ± 2‰ were determined under steady conditions. As ΛC/Cl values are independent of complicating masking effects, and thus reflect reaction mechanisms, dual C-Cl isotope plots suggested distinct DCM degradation pathways under steady and transient conditions with ΛC/Cl values of 1.68 ± 0.26 and 3.41 ± 0.50, respectively. Even though a contribution of different mechanisms may take place during transient conditions, ΛC/Cl values fall in the range of SN1 pathways reported for Ca. Dichloromethanomonas elyunquensis (ΛC/Cl = 3.40 ± 0.03). The distinct ΛC/Cl values may imply mechanistically distinct C-Cl bond cleavage reactions subjected to microbial adaptations during dynamic hydrogeological conditions. Although bacterial communities did not significantly change over time, the occurrence of Geobacter under both steady and transient conditions supports DCM degradation under iron-reducing prevailing conditions. Altogether, our results highlight that water table fluctuations enhance DCM biodegradation and influence DCM degradation pathways compared to steady conditions. This integrative study provides new insights into in situ degradation of DCM in contaminated aquifers and accounts the effects of dynamic water tables on DCM degradation.

Reactive transport in porous media involves a complex interplay of multiple processes relative to flow of water and gases, transport of elements, chemical reactions and microbial activities. In surface-groundwater interfaces, the role of the capillary fringe is of particular interest as water table variations can strongly impact the transfer of gases (e.g. oxygen), the evolution of redox conditions and the evolution/adaptation of bacterial/microbial populations that control biodegradation pathways of contaminants. Although the understanding of individual processes is advanced, their interactions are not yet fully understood challenging the development of efficient reactive transport models (RTM) for predictive applications. In this context, the combination of microbial approaches with isotope measurements and modelling may be useful to understand reactive transport of halogenated pollutants in hydrogeological dynamic systems, to improve processes representation in RTMs, and to reduce model equifinality. Dichloromethane (DCM) is a toxic and volatile halogenated compound frequently detected in multi-contaminated aquifers. Although mechanisms of DCM microbial degradation under both aerobic and anaerobic conditions have been described, little is known about the relationships between the hydrogeochemical variations caused by water table fluctuations, as well as their effects on the diversity and distribution of bacterial communities and degradation pathways. In this study, two laboratory aquifers fed by contaminated groundwater from the industrial site Thermeroil (France) were designed to collect water samples at high-resolution to investigate the reactive transport of DCM in porous media under steady and dynamic hydrogeological conditions. The effect of water table variations on hydrochemical, microbial and isotopic composition (δ13C and δ37Cl) was examined to derive DCM mass removal and potential changes in degradation pathways. For the latter, Compound-Stable Isotope Analysis (CSIA) has been used as a tool to evaluate natural degradation of halogenated hydrocarbons. A RTM model (CubicM) is currently being developed to include dual-element CSIA and biological processes - such as growth, decay, attachment, detachment or dormancy – and relate changes in redox conditions with the evolution of DCM degrading populations. A two-phase flow model (i.e. water and gas) has been developed to account for the volatilization and the associated transport processes of halogenated volatile compounds in porous media. Currently, the model is tested on the experimental results to assist in the interpretation of DCM dissipation and the observed biogeochemical and microbial processes to determine the best-suited formalism to address the effect of water table fluctuations on DCM reactive transport in porous media. Such model will enable to assess natural attenuation of DCM at contaminated sites accounting for dynamic hydrogeological conditions.

Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization and regulation of such gene clusters is highlighted for representative organohalide-degrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides.

Chlorinated ethenes (CEs) are most problematic pollutants in groundwater. Dehalogenating bacteria, and in particular organohalide-respiring bacteria (OHRB), can transform PCE to ethene under anaerobic conditions, and thus contribute to bioremediation of contaminated sites. Current approaches to characterize in situ biodegradation of CEs include hydrochemical analyses, quantification of the abundance of key species (e.g. Dehalococcoides mccartyi) and dehalogenase genes (pceA, vcrA, bvcA and tceA) involved in different steps of organohalide respiration (OHR) by qPCR, and compound-specific isotope analysis (CSIA) of CEs. Here we combined these approaches with sequencing of 16S rRNA gene amplicons to consider both OHRB and bacterial taxa involved in CE transformation at a multi-contaminated site. Integrated analysis of hydrogeochemical characteristics, gene abundances and bacterial diversity shows that bacterial diversity and OHRB mainly correlated with hydrogeochemical conditions, suggesting that pollutant exposure acts as a central driver of bacterial diversity. CSIA, abundances of four reductive dehalogenase encoding genes and the prevalence of Dehalococcoides highlighted sustained PCE, DCE and VC degradation in several wells of the polluted plume. These results suggest that bacterial taxa associated with OHR play an essential role in natural attenuation of CEs, and that representatives of taxa including Dehalobacterium and Desulfosporosinus co-occur with Dehalococcoides. Overall, our study emphasizes the benefits of combining several approaches to evaluate the interplay between the dynamics of bacterial diversity in CE-polluted plumes and in situ degradation of CEs, and to contribute to a more robust assessment of natural attenuation at multi-polluted sites.

International audience; Methylobacterium strains can use one-carbon compounds, such as methanol, for methylotrophic growth. In addition to methanol, a few strains also utilize dichloromethane, a major industrial chlorinated solvent pollutant. With a fully assembled and annotated genome, M. extorquens DM4 is the reference bacterium for aerobic dichloromethane degradation. The doublet N-terminal oriented proteomics (dN-TOP) strategy was applied to further improve its genome annotation and a differential proteomics approach was performed to compare M. extorquens DM4 grown either with methanol or dichloromethane as the sole source of carbon and energy. These approaches led to experimental confirmation of 259 hypothetical proteins, correction of 78 erroneous predicted start codons, discovery of 39 new proteins and annotation of 66 signal peptides, including essential enzymes involved in methylotrophic growth.SignificanceDichloromethane (methylene chloride, CH2Cl2, DCM) is one of the most widely used industrial halogenated solvents and a potential carcinogen. Microbial rehabilitation of worldwide-contaminated sites involves DCM breakdown by bacteria that are able to grow using this pollutant as their sole carbon and energy source. The most-studied methylotrophic DCM degrader is Methylobacterium extorquens strain DM4. Proteomic studies of the Methylobacterium genus have been performed previously, but genome-wide investigation of N-termini of expressed proteins has not yet been performed. Differential quantitative proteomic analysis also opens new research perspectives to better monitor and understand bacterial growth with DCM.

German Science Foundation (DFG KE 884/10-1 ; ANR-14-CE35-0005-01) ; French National Research Agency (DFG KE 884/10-1 ; ANR-14-CE35-0005-01); International audience; Chloromethane (CH3Cl, methyl chloride) is the most abundant volatile halocarbon in the atmosphere and involved in stratospheric ozone depletion. The global CH3Cl budget, and especially the CH3Cl sink from microbial degradation in soil, still involves large uncertainties. These may potentially be resolved by a combination of stable isotope analysis and bacterial diversity studies. We determined the stable isotope fractionation of CH3Cl hydrogen and carbon and investigated bacterial diversity during CH3Cl degradation in three soils with different properties (forest, grassland, and agricultural soils) and at different temperatures and headspace mixing ratios of CH3Cl. The extent of chloromethane degradation decreased in the order forest > grassland > agricultural soil. Rates ranged from 0.7 to 2.5 μg g−1 dry wt. d−1 for forest soil, from 0.1 to 0.9 μg g−1 dry wt. d−1 for grassland soil, and from 0.1 to 0.4 μg g−1 dry wt. d−1 for agricultural soil and increased with increasing temperature and CH3Cl supplementation. The measured mean stable hydrogen enrichment factor of CH3Cl of −50 ± 13‰ was unaffected by temperature, mixing ratio, or soil type. In contrast, the stable carbon enrichment factor depended on CH3Cl degradation rates and ranged from −38 to −11‰. Bacterial community composition correlated with soil properties was independent from CH3Cl degradation or isotope enrichment. Nevertheless, increased abundance after CH3Cl incubation was observed in 21 bacterial operational taxonomical units (OTUs at the 97% 16S RNA sequence identity level). This suggests that some of these bacterial taxa, although not previously associated with CH3Cl degradation, may play a role in the microbial CH3Cl sink in soil.

International audience; Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.

L’utilisation intensive des hydrocarbures chlorés aliphatiques (tels que le tétrachloroéthylène (PCE) et ses produits de dégradation) a entraîné leur accumulation et leur persistance dans les aquifères du monde entier, représentant un risque sérieux pour la santé humaine et l'environnement. Leur (bio)remédiation nécessite en amont une caractérisation approfondie du panache de pollution. Or, bien qu’une caractérisation physico-chimique s’avère indispensable, une évaluation du potentiel de biodégradation àl’aide d’outils de biologie moléculaire et d’isotopie peut apporter une plus-value au diagnostic du site. La présente étude avait pour objectif d’évaluer si, dans une nappe contaminée par des éthènes chlorés (PCE, TCE, DCE et CV), une biodégradation a lieu et peut être reliée à l’abondance de certains gènes fonctionnels impliqués dans la déchloration réductrice et à une diversité bactérienne spécifique. In fine, la plus-value de ces approches moléculaires ainsi que leur combinaison avec l’isotopie composé spécifique est évaluée.Douze piézomètres, répartis au niveau de la source de la pollution (usine), du panache et de la frange, ont été suivis tous les 6 mois pendant 2 ans sur un ancien site industriel contaminé, et des analyses de quatre types ont été effectuées : i) caractérisation physico-chimique des eaux, ii) isotopie composéspécifique, iii) abondance de gènes fonctionnels spécifiques, et iv) analyse métagénomique de labiodiversité bactérienne. En plus de cette caractérisation in situ, des mesures de potentiels de dégradation des éthènes chlorés ont été réalisées en batch.Le suivi temporel et spatial (gènes fonctionnels et métagénomique) a permis de confirmer la présence de bactéries capables de dégrader les polluants, leur abondance (PCR quantitative), la présence d’une biodégradation in situ et son ampleur (isotopie composéspécifique ou CSIA). Ces résultats mettent en évidence la plus-value de la caractérisation biologique des sites pollués pour guider les stratégies de remédiation et la gestion du site.

The aim of this work was to develop a continuous liquid/gas membrane bioreactor (L/G MBR), i.e. a fermenting module including hollow fibers membrane for L/G separation, for biohydrogen production by dark fermentation. Originally seeded with sludge from a wastewater treatment plant, the L/G MBR underwent a complete stop for eight months. It was then operated without further reseeding. In the present experiment, performed 551 days after the last reseeding, average hydrogen yield of 1.1 ± 0.2 mol per mol glucose added and hydrogen productivity of 135 ± 22 mL/L/h were reached, with acetate and butyrate as the main metabolite products. DNA sequence analysis revealed that Clostridium beijerinckii, Clostridium pasteurianum and Enterobacter sp. were dominant in liquid outlet, in a biofilm on the surface of the hollow fibers and in microbial granules. The L/G MBR has potential for the concentration and the long-term maintenance of an active hydrogen-producing bacterial community without need for reseeding.

SUMMARY Methanobactins (mbs) are low-molecular-mass (2+ to Cu 1+ . In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs.

Dichloromethane (DCM) is a widespread and toxic industrial solvent which often co-occurs with chlorinated ethenes at polluted sites. Biodegradation of DCM occurs under both oxic and anoxic conditions in soils and aquifers. Here we investigated in situ and ex situ biodegradation of DCM in groundwater sampled from the industrial site of Themeroil (France), where DCM occurs as a major co-contaminant of chloroethenes. Carbon isotopic fractionation (eC) for DCM ranging from −46 to −22‰ were obtained under oxic or denitrifying conditions, in mineral medium or contaminated groundwater, and for laboratory cultures of Hyphomicrobium sp. strain GJ21 and two new DCM-degrading strains isolated from the contaminated groundwater. The extent of DCM biodegradation (B%) in the aquifer, as evaluated by compound-specific isotope analysis (δ13C), ranged from 1% to 85% applying DCM-specific eC derived from reference strains and those determined in this study. Laboratory groundwater microcosms under oxic conditions showed DCM biodegradation rates of up to 0.1 mM·day−1, with concomitant chloride release. Dehalogenase genes dcmA and dhlA involved in DCM biodegradation ranged from below 4 × 102 (boundary) to 1 × 107 (source zone) copies L−1 across the contamination plume. High-throughput sequencing on the 16S rrnA gene in groundwater samples showed that both contaminant level and terminal electron acceptor processes (TEAPs) influenced the distribution of genus-level taxa associated with DCM biodegradation. Taken together, our results demonstrate the potential of DCM biodegradation in multi-contaminated groundwater. This integrative approach may be applied to contaminated aquifers in the future, in order to identify microbial taxa and pathways associated with DCM biodegradation in relation to redox conditions and co-contamination levels.

Halogenated volatile organic compounds (VOCs) emitted by terrestrial ecosystems, such as chloromethane (CH3Cl), have pronounced effects on troposphere and stratosphere chemistry and climate. The magnitude of the global CH3Cl sink is uncertain since it involves a largely uncharacterized microbial sink. CH3Cl represents a growth substrate for some specialized methylotrophs, while methanol (CH3OH), formed in much larger amounts in terrestrial environments, may be more widely used by such microorganisms. Direct measurements of CH3Cl degradation rates in two field campaigns and in microcosms allowed the identification of top soil horizons (i.e., organic plus mineral A horizon) as the major biotic sink in a deciduous forest. Metabolically active members of Alphaproteobacteria and Actinobacteria were identified by taxonomic and functional gene biomarkers following stable isotope labeling (SIP) of microcosms with CH3Cl and CH3OH, added alone or together as the [C-13]-isotopologue. Well-studied reference CH3Cl degraders, such as Methylobacterium extorquens CM4, were not involved in the sink activity of the studied soil. Nonetheless, only sequences of the cmuA chloromethane dehalogenase gene highly similar to those of known strains were detected, suggesting the relevance of horizontal gene transfer for CH3Cl degradation in forest soil. Further, CH3Cl consumption rate increased in the presence of CH3OH. Members of Alphaproteobacteria and Actinobacteria were also C-13-labeled upon [C-13]-CH3OH amendment. These findings suggest that key bacterial CH3Cl degraders in forest soil benefit from CH3OH as an alternative substrate. For soil CH3Cl-utilizing methylotrophs, utilization of several one-carbon compounds may represent a competitive advantage over heterotrophs that cannot utilize one-carbon compounds.

Common herbicides such as metolachlor (MET), and their transformation products, are frequently detected in groundwater worldwide. Little is known about the response of groundwater bacterial communities to herbicide exposure, and its potential use for ecotoxicological assessment. The response of bacterial communities exposed to different levels of MET from the Ariege alluvial aquifer (Southwest of France) was investigated in situ and in laboratory experiments. Variations in both chemistry and bacterial communities were observed in groundwater, but T-RFLP analysis did not allow to uncover a pesticide-specific effect on endogenous bacterial communities. To circumvent issues of hydrogeochemical and seasonal variations in situ, groundwater samples from two monitoring wells of the Ariege aquifer with contrasting records of pesticide contamination were exposed to different levels of MET in laboratory experiments. The standard Microtox® acute toxicity assay did not indicate toxic effects of MET, even at 5 mg L-1 (i.e., 1000-fold higher than in contaminated groundwater). Analysis of MET transformation products and compound-specific isotope analysis (CSIA) in laboratory experiments demonstrated MET biodegradation but did not correlate with MET exposure. High-throughput sequencing analysis (Illumina MiSeq) of bacterial communities based on amplicons of the 16S rRNA gene revealed that bacterial community differed mainly by groundwater origin rather than by its response to MET exposure. OTUs correlating with MET addition ranged between 0.4 to 3.6% of the total. Predictive analysis of bacterial functions impacted by pesticides using PICRUSt suggested only minor changes in bacterial functions with increasing MET exposure. Taken together, results highlight MET biodegradation in groundwater, and the potential use of bacterial communities as sensitive indicators of herbicide contamination in aquifers. Although detected effects of MET on groundwater bacterial communities were modest, this study illustrates the potential of integrating DNA- and isotopic analysis-based approaches to improve ecotoxicological assessment of pesticide-contaminated aquifers. Open in a separate window GRAPHICAL ABSTRACT An integrative approach was develop to investigate in situ and in laboratory experiments the response of bacterial communities exposed to different levels of MET from the Ariege alluvial aquifer (Southwest of France).

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.

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Stormwater wetlands collect and attenuate runoff-related herbicides, limiting their transport into aquatic ecosystems. Knowledge on wetland bacterial communities with respect to herbicide dissipation is scarce. Previous studies showed that hydrological and hydrochemical conditions, including pesticide removal capacity, may change from spring to summer in stormwater wetlands. We hypothesized that these changes alter bacterial communities, which, in turn, influence pesticide degradation capacities in stormwater wetland. Here, we report on bacterial community changes in a stormwater wetland exposed to pesticide runoff, and the occurrence of trz, atz, puh, and phn genes potentially involved in the biodegradation of simazine, diuron, and glyphosate. Based on T-RFLP analysis of amplified 16S rRNA genes, a response of bacterial communities to pesticide exposure was not detected. Changes in stormwater wetland bacterial community mainly followed seasonal variations in the wetland. Hydrological and hydrochemical fluctuations and vegetation development in the wetland presumably contributed to prevent detection of effects of pesticide exposure on overall bacterial community. End point PCR assays for trz, atz, phn, and puh genes associated with herbicide degradation were positive for several environmental samples, which suggest that microbial degradation contributes to pesticide dissipation. However, a correlation of corresponding genes with herbicide concentrations could not be detected. Overall, this study represents a first step to identify changes in bacterial community associated with the presence of pesticides and their degradation in stormwater wetland.

The fractionation of carbon and chlorine stable isotopes of dichloromethane (CH2Cl2, DCM) upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed. Isotope effects for individual steps of the multi-stage DCM degradation process, including transfer across the cell wall from the aqueous medium to the cell cytoplasm, dehalogenase binding, and catalytic reaction, were considered. The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of CCl bonds, and not by inflow of DCM into cells. Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures, presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess. Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes (Δδ13C/Δδ37Cl), increasing corresponding ratio from 7.2-8.6 to 9.6-10.5 under conditions of glutathione deficiency. This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM, unlike the uncatalyzed reaction, which is a one-stage process, as shown by quantum-chemical modeling.

Engineering organisms for biotechnology applications requires knowledge of their essential genes and associated regulatory networks. A new study of methylotrophic metabolism in Methylobacterium reveals essentiality of the unregulated, off-pathway phosphoribulokinase gene and an unexpected key regulatory role for its product ribulose-1,5-bisphosphate.

The genome sequence of Hyphomicrobium sp. strain GJ21, isolated in the Netherlands from samples of environments contaminated with halogenated pollutants and capable of using dichloromethane as its sole carbon and energy source, was determined.

The genomes of the aerobic methanotrophs “ Methyloterricola oryzae ” strain 73a T and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73a T represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum .

Knowledge of wetland bacterial communities in the context of pesticide contamination and hydrological regime is scarce. We investigated the bacterial composition in constructed wetlands receiving Mercantor Gold ® contaminated water (960 g L − 1 of the herbicide S -metolachlor, > 80% of the S -enantiomer) operated under continuous-flow or batch modes to evaluate the impact of the hydraulic regime. In the continuous-flow wetland, S -metolachlor mass removal was > 40%, whereas in the batch wetland, almost complete removal of S -metolachlor (93–97%) was observed. Detection of ethanesulfonic and oxanilic acid degradation products further indicated S -metolachlor biodegradation in the two wetlands. The dominant bacterial populations were characterised by terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing. The bacterial profiles evolved during the first 35 days of the experiment, starting from a composition similar to that of inlet water, with the use of nitrate and to a lesser extent sulphate and manganese as terminal electron acceptors for microbial metabolism. Proteobacteria were the most abundant phylum, with Beta -, Alpha - and Gammaproteobacteria representing 26%, 19% and 17% respectively of total bacterial abundance. Bacterial composition in wetland water changed gradually over time in continuous-flow wetland and more abruptly in the batch wetland. Differences in overall bacterial water structure in the two systems were modest but significant ( p = 0.008), and S -metolachlor, nitrate, and total inorganic carbon concentrations correlated with changes in the bacterial profiles. Together, the results highlight that bacterial composition profiles and their dynamics may be used as bioindicators of herbicide exposure and hydraulic disturbances in wetland systems.