Diversity of novel archaeal viruses infecting methanogens discovered through coupling of stable isotope probing and metagenomics.

Diversity of viruses infecting non-extremophilic archaea has been grossly understudied. This is particularly the case for viruses infecting methanogenic archaea, key players in the global carbon biogeochemical cycle. Only a dozen of methanogenic archaeal viruses have been isolated so far. In the present study, we implemented an original coupling between stable isotope probing and complementary shotgun metagenomic analyses to identify viruses of methanogens involved in the bioconversion of formate, which was used as the sole carbon source in batch anaerobic digestion microcosms. Under our experimental conditions, the microcosms were dominated by methanogens belonging to the order Methanobacteriales (Methanobacterium and Methanobrevibacter genera). Metagenomic analyses yielded several previously uncharacterized viral genomes, including a complete genome of a head-tailed virus (class Caudoviricetes, proposed family Speroviridae, Methanobacterium host) and several near-complete genomes of spindle-shaped viruses. The two groups of viruses are predicted to infect methanogens of the Methanobacterium and Methanosarcina genera and represent two new virus families. The metagenomics results are in good agreement with the electron microscopy observations, which revealed the dominance of head-tailed virus-like particles and the presence of spindle-shaped particles. The present study significantly expands the knowledge on the viral diversity of viruses of methanogens.

Asymmetrical response of anaerobic digestion microbiota to temperature changes.

In natural settings, anaerobic digestion can take place in a wide temperature range, but industrial digesters are usually operated under either mesophilic (~35 °C) or thermophilic (~55 °C) conditions. The ability of anaerobic digestion microbiota to switch from one operating temperature to the other remains poorly documented. We therefore studied the effect of sudden temperature changes (35 °C/55 °C) in lab-scale bioreactors degrading 13C-labelled cellulose. An asymmetric behaviour was observed. In terms of methane production, after an adaptation period, mesophilic inoculum exhibited a functional resistance to temperature increase but no functional resilience when temperature was reset to 35 °C, while thermophilic inoculum methanogenic activity strongly decreased under mesophilic conditions but partially recovered when temperature was reset to 55 °C. Automated ribosomal intergenic spacer analysis community fingerprints evidenced a strong influence of temperature on microbial diversity, particularly pronounced and persistent for Archaea. Key phylotypes involved in 13C-cellulose degradation were identified with a coupled stable isotope probing (SIP)-16S rDNA pyrotag sequencing approach, suggesting that the hydrolytic and fermentative metabolic functions could be maintained thanks to functional redundancy between members of the class Clostridia, whereas methanogenic activity primarily relied on specialized groups affiliated either to genus Methanosarcina (mesophilic conditions), Methanothermobacter or Methanoculleus (thermophilic conditions) that were irreversibly modified by temperature increase.

Increasing concentrations of phenol progressively affect anaerobic digestion of cellulose and associated microbial communities.

Performance stability is a key issue when managing anaerobic digesters. However it can be affected by external disturbances caused by micropollutants. In this study the influence of phenol on the methanization of cellulose was evaluated through batch toxicity assays. Special attention was given to the dynamics of microbial communities by means of automated ribosomal intergenic spacer analysis. We observed that, as phenol concentrations increased, the different steps of anaerobic cellulose digestion were unevenly and progressively affected, methanogenesis being the most sensitive: specific methanogenic activity was half-inhibited at 1.40 g/L of phenol, whereas hydrolysis of cellulose and its fermentation to VFA were observed at up to 2.00 g/L. Depending on the level of phenol, microbial communities resisted either through physiological or structural adaptation. Thus, performances at 0.50 g/L were maintained in spite of the microbial community's shift. However, the communities' ability to adapt was limited and performances decreased drastically beyond 2.00 g/L of phenol.

Penta- and 2,4,6-tri-chlorophenol biodegradation during municipal solid waste anaerobic digestion.

In this study isotopic tracing using (13)C labelled pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP) is proposed as a tool to distinguish the loss of PCP and 2,4,6-TCP due to biodegradation from other physical processes. This isotopic approach was applied to accurately assess in situ PCP and 2,4,6-TCP degradation under methanogenic conditions in several microcosms made up of household waste. These microcosms were incubated in anaerobic conditions at 35°C (mesophilic) and 55°C (thermophilic) without agitation. The volume of biogas produced (CH4 and CO2), was followed for a period of 130 days. At this stage of stable methanogenesis, (13)C6-PCP and (13)C6-2,4,6-TCP were introduced anaerobically in microcosms and its monitoring at mesophilic and thermophilic conditions was performed in parallel by gas chromatography mass spectrometry (GC-MS) and gas chromatography isotope-ratio mass spectrometry (GC-IRMS). This study proved the almost total dechlorination of bioavailable PCP and 2,4,6-TCP into 4-CP at 35°C. Nevertheless, high rate adsorption in particular materials of the two compounds was observed. Furthermore, Carbon-13 Nuclear Magnetic Resonance ((13)C-NMR) Spectroscopy analysis of (13)C labelled 2,4,6-TCP mesophilic incubations showed the partial mineralization of 4-CP at 35°C to acetate and then to HCO(3-). Consequently, NMR results confirm the biogas isotopic results indicating the mineralization of (13)C labelled 2,4,6-TCP into (13)C (CH4 and CO2). Concerning (13)C labelled PCP mesophilic incubations, the isotopic composition of the biogas still natural until the day 262. In contrast, no dechlorination was observed at 55°C. Thus PCP and 2,4,6-TCP were persistent in thermophilic conditions.

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 PXD002420 (http://proteomecentral.proteomexchange.org/dataset/PXD002420).

Occurrence of lignin degradation genotypes and phenotypes among prokaryotes.

A number of prokaryotes actively contribute to lignin degradation in nature and their activity could be of interest for many applications including the production of biogas/biofuel from lignocellulosic biomass and biopulping. This review compares the reliability and efficiency of the culture-dependent screening methods currently used for the isolation of ligninolytic prokaryotes. Isolated prokaryotes exhibiting lignin-degrading potential are presented according to their phylogenetic groups. With the development of bioinformatics, culture-independent techniques are emerging that allow larger-scale data mining for ligninolytic prokaryotic functions but today, these techniques still have some limits. In this work, two phylogenetic affiliations of isolated prokaryotes exhibiting ligninolytic potential and laccase-encoding prokaryotes were determined on the basis of 16S rDNA sequences, providing a comparative view of results obtained by the two types of screening techniques. The combination of laboratory culture and bioinformatics approaches is a promising way to explore lignin-degrading prokaryotes.

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.

Data on the influence of temperature on the growth of Escherichia coli in a minimal medium containing glucose as the sole carbon source for the joint computation of growth yields and rates at each temperature from 27 to 45°C.

Temperature is a key factor influencing microbial growth rates and yields. In literature, the influence of temperature on growth is studied either on yields or rates but not both at the same time. Moreover, studies often report the influence of a specific set of temperatures using rich culture media containing complex ingredients (such as yeast extract) which chemical composition cannot be precisely specified. Here, we present a complete dataset for the growth of Escherichia coli K12 NCM3722 strain in a minimal medium containing glucose as the sole energy and carbon source for the computation of growth yields and rates at each temperature from 27 to 45°C. For this purpose, we monitored the growth of E. coli by automated optical density (OD) measurements in a thermostated microplate reader. At each temperature full OD curves were reported for 28 to 40 microbial cultures growing in parallel wells. Additionally, a correlation was established between OD values and the dry mass of E. coli cultures. For that, 21 dilutions were prepared from triplicate cultures and optical density was measured in parallel with the microplate reader (ODmicroplate) and a UV-Vis spectrophotometer (ODUV-vis) and correlated to duplicate dry biomass measurements. The correlation was used to compute growth yields in terms of dry biomass.

Systematic and quantitative analysis of two decades of anodic wastewater treatment in bioelectrochemical reactors.

Wastewater treatment is generally performed using energy-intensive processes, such as activated sludge. Improving energy efficiency has become one of the main challenges for next-generation wastewater treatment plants. Bioelectrochemical systems (BES) have been attracting attention because they take advantage of the chemical energy contained in wastewater while enabling the valorization of effluents: either with electrical energy (microbial fuel cells) or with useful chemicals (microbial electrolysis cells). Bioelectrochemical wastewater treatment has been under investigation since the early 2000s and is now the subject of an abundant literature, which is most frequently focused on anodic COD removal. Comparing results obtained in different studies is particularly difficult with BES, because many different parameters (effluent characteristics, inoculation, design, and operation) may interact and because using real effluents results in high variability. To address this issue, data were retrieved from 1,073 articles that were selected objectively and with transparency. This systematic review evaluates the potential of anodic wastewater treatment, based on 4,579 experimental observations. Overall, BES has already shown satisfactory treatment capacity, with a median chemical oxygen demand removal of 72%. However, the median coulombic efficiency was only 18%, increasing this parameter offers the greatest opportunity for BES improvement.

Gradual development of ammonia-induced syntrophic acetate-oxidizing activities under mesophilic and thermophilic conditions quantitatively tracked using multiple isotopic approaches.

Insights into microbiota adaptation to increased ammonia stress, and identification of indicator microorganisms can help to optimize the operation of anaerobic digesters. To identify microbial indicators and investigate their metabolic contribution to acetoclastic methanogenesis (AM), syntrophic acetate oxidation (SAO) or hydrogenotrophic methanogenesis (HM), 40 anaerobic batch reactors fed with acetate of 110 mmol/L were set up at NH4+-N concentrations of 0.14 g/L, 5.00 g/L or 7.00 g/L, inoculated with thermophilic or mesophilic microbiota with or without pre-exposure to ammonia stress. Four stable carbon isotope probing approaches were applied in parallel, with [1,2-13C]-CH3COOH, [2-13C]-CH3COOH, [13C]NaHCO3 or non-labeled CH3COOH used individually. The last three approaches were used to quantify the methanogenic pathways by tracking labeled 13C or natural 13C signatures in the resulting CH4 and CO2, and consistently detected the dynamic transition of dominant pathways from AM to SAO-HM under ammonia stress. Results of quantitative PCR and fluorescence in-situ hybridization illustrated the procedure, acetotrophic methanogens being outcompeted by acetate-oxidizing syntrophs. The first and last isotope-labeling approaches were designed to probe the active acetate-mineralizing microbes with DNA-SIP. Known acetate-oxidizing bacteria like Syntrophaceticus and Tepidanaerobacter, as well as novel members of Pseudomonas, Bacillus and Symbiobacteraceae were detected, with Methanoculleus as the predominant H2/CO2-utilizing partner. Using NanoSIMS, some bacterial cells were observed to be fixing CO2 from [13C]NaHCO3. In this study, Methanosaeta was only active with ammonia < 200 mg-N/L; the syntrophs catalyzing SAO-HM started to compete with AM-conducting Methanosarcina at intermediate concentrations of ammonia, i.e. 200-500 mg-N/L, and outcompeted the acetotrophic methanogens with ammonia > 500 mg-N/L. Under ammonia stress, diverse known and novel microbial taxa were involved in acetate mineralization, comparable with those identified in previous studies.

Denitrifying bio-cathodes developed from constructed wetland sediments exhibit electroactive nitrate reducing biofilms dominated by the genera Azoarcus and Pontibacter.

To limit the nitrate contamination of ground and surface water, stimulation of denitrification by electrochemical approach is an innovative way to be explored. Two nitrate reducing bio-cathodes were developed under constant polarization (-0.5 V vs SCE) using sediments and water from a constructed wetland (Rampillon, Seine-et-Marne, France). The bio-cathodes responded to nitrate addition on chronoamperometry through an increase of the reductive current. The denitrification efficiency of the pilots increased by 47% compared to the negative controls without electrodes after polarization. 16S rRNA gene sequencing of the biofilms and sediments evidenced the significant and discriminating presence of the Azoarcus and Pontibacter genera in the biofilms from biocathodes active for nitrate reduction. Our study shows the possibility to promote the development of efficient Azoarcus-dominated biocathodes from freshwater sediment to enhance nitrate removal from surface waters.

Electrochemical analysis of a microbial electrochemical snorkel in laboratory and constructed wetlands.

Microbial electrochemical snorkel (MES) is a short-circuited microbial fuel cell applicable to water treatment that does not produce energy but requires lower cost for its implementation. Few reports have already described its water treatment capabilities but no deeper electrochemical analysis were yet performed. We tested various materials (iron, stainless steel and porous graphite) and configurations of snorkel in order to better understand the rules that will control in a wetland the mixed potential of this self-powered system. We designed a model snorkel that was studied in laboratory and on the field. We confirmed the development of MES by identifying anodic and cathodic parts, by measuring the current between them and by analyzing microbial ecology in laboratory and field experiments. An important application is denitrification of surface water. Here we discuss the influence of nitrate on its electrochemical response and denitrification performances. Introducing nitrate caused the increase of the mixed potential of MES and of current at a potential value relatively more positive than for nitrate-reducing biocathodes described in the literature. The major criteria for promoting application of MES in artificial wetland dedicated to mitigation of non-point source nitrate pollution from agricultural water are considered.

Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate.

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.

Insights from Microbial Transition State Theory on Monod's Affinity Constant.

Microbial transition state theory (MTS) offers a theoretically explicit mathematical model for substrate limited microbial growth. By considering a first order approximation of the MTS equation one recovers the well-known Monod's expression for growth, which was regarded as a purely empirical function. The harvest volume of a cell as defined in MTS theory can then be related to the affinity concept, giving a new physical interpretation to it, and a new way to determine its value. Consequences of such a relationship are discussed.

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

DNA-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.

Consistent microbial dynamics and functional community patterns derived from first principles.

Microbial communities are key engines that drive earth's biogeochemical cycles. However, existing ecosystem models have only limited ability to predict microbial dynamics and require the calibration of multiple population-specific empirical equations. In contrast, we build on a new kinetic "Microbial Transition State" (MTS) theory of growth derived from first principles. We show how the theory coupled to simple mass and energy balance calculations provides a framework with intrinsically important qualitative properties to model microbial community dynamics. We first show how the theory can simultaneously account for the influence of all the resources needed for growth (electron donor, acceptor, and nutrients) while still producing consistent dynamics that fulfill the Liebig rule of a single limiting substrate. We also show consistent patterns of energy-dependent microbial successions in mixed culture without the need for calibration of population-specific parameters. We then show how this approach can be used to model a simplified activated sludge community. To this end, we compare MTS-derived dynamics with those of a widely used activated sludge model and show that similar growth yields and overall dynamics can be obtained using two parameters instead of twelve. This new kinetic theory of growth grounded by a set of generic physical principles parsimoniously gives rise to consistent microbial population and community dynamics, thereby paving the way for the development of a new class of more predictive microbial ecosystem models.

Biorefinery for heterogeneous organic waste using microbial electrochemical technology.

Environmental biorefineries aim to produce biofuels and platform biomolecules from organic waste. To this end, microbial electrochemical technologies theoretically allow controlled microbial electrosynthesis (MES) of organic molecules to be coupled to oxidation of waste. Here, we provide a first proof of concept and a robust operation strategy for MES in a microbial electrolysis cell (MEC) fed with biowaste hydrolysates. This strategy allowed stable operation at 5 A/m2 for more than three months in a labscale reactor. We report a two to four-fold reduction in power consumption compared to microbial electrosynthesis with water oxidation at the anode. The bioelectrochemical characterizations of the cells were used to compute energy and matter balances for biorefinery scenarios in which anaerobic digestion (AD) provides the electricity and CO2 required for the MEC. Calculations shows that up to 22% of electrons (or COD) from waste may be converted to organic products in the AD-MEC process.

Comparative metatranscriptomic analysis of anaerobic digesters treating anionic surfactant contaminated wastewater.

Three distinct biological reactors fed with synthetic medium (UASB_Control), synthetic medium and linear alkylbenzene sulfonate (LAS; UASB_SL), and real laundry wastewater (UASB_LW) were compared using a metatranscriptomic approach to determine putative bioindicator genes and taxonomies associated to all steps of anaerobic LAS biodegradation pathway. A homemade bioinformatics pipeline combined with an R workflow was developed to perform the RNAseq data analysis. UASB_SL and UASB_LW showed similar values of LAS biological degradation (~47%) and removal (53-55%). Rarefaction analysis revealed that 1-2 million reads were sufficient to access the whole functional capacity. In the first step of LAS biodegradation pathway, fumarate reductase subunit C was detected and taxonomically assigned to the genus Syntrophobacter (0.002% - UASB_SL; 0.0015% - UASB_LW; not detected - UASB_Control). In the second step, many enzymes related to beta-oxidation were observed and most of them with low relative abundance in UASB Control and taxonomically related with Smithella, Acinetobacter and Syntrophorhabdus. For the ring cleavage step, the abundance of 6 OCH CoA hydrolase putative gene was ten times higher in UASB_SL and UASB_LW when compared to UASB_Control, and assigned to Desulfomonile and Syntrophorhabdus. Finally, the adenylylsulfate reductase, taxonomically related with Desulfovibrio and Desulfomonile, was observed in the desulfonation step with the highest relative abundance in UASB_LW.

Simultaneous analysis of microbial identity and function using NanoSIMS.

Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of 13C or 15N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated.

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach.

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.