Microbial and functional characterization of granulated sludge from full-scale UASB thermophilic reactor applied to sugarcane vinasse treatment.

Considering the scarcity of data in the literature regarding phylogenetic and metabolic composition of different inocula, especially those from thermophilic conditions, this research aimed at characterizing the microbial community and preferable metabolic pathways of an UASB reactor sludge applied to the thermophilic treatment (55°C) of sugarcane vinasse, by means of shotgun metagenomics. After its metabolic potential was depicted, it was possible to observe several genes encoding enzymes that are of great importance to anaerobic digestion processes with different wastes as substrate, especially regarding the biodegradation of carbohydrates and ligninolytic compounds, glycerolypids, volatile fatty acids and alcohols metabolism and biogas (H2 and CH4) production. The genera identified in higher relative abundances for Bacteria domain were Sulfirimonas (37.52 ± 1.8%), possibly related to the sludge endogenic activity due to its strong relation with a peptidoglycan lyase enzymes family, followed by Fluviicola (5.01 ± 1.0%), Defluviitoga (4.36 ± 0.2%), Coprothermobacter (4.32 ± 0.5%), Fervidobacterium (2.93 ± 0.3%), Marinospirillum (2.75 ± 0.2%), Pseudomonas (2.14 ± 0.2%) and Flavobacterium (1.78 ± 0.1%), mostly related with carbohydrates fermentations and/or H2 production. For Archaea domain, Methanosarcina (0.61 ± 0.1%), Methanothermobacter (0.38 ± 0.0%), Methanoculleus (0.30 ± 0.1%), Thermococcus (0.03 ± 0.0%), Methanolobus (0.02 ± 1.8%), Methanobacterium (0.013 ± 0.0%), Aciduliprofundum and Pyrococcus (0.01 ± 0.0%) were the most dominant ones, being Methanosarcina the most related with methanogenesis. It was concluded that the robust inoculum description performed in this study may subside future biotechnological researches by using similar inocula (UASB sludges), focusing on the obtainment of value-added by-products by means of anaerobic digestion, such as volatile fatty acids, alcohols and biogas (H2 and CH4), by using several types of waste as substrate.

Wild type and variants of SARS-COV-2 in Parisian sewage: presence in raw water and through processes in wastewater treatment plants.

The presence of SARS-CoV-2 RNA has been extensively reported at the influent of wastewater treatment plants (WWTPs) worldwide, and its monitoring has been proposed as a potential surveillance tool to early alert of epidemic outbreaks. However, the fate of the SARS-CoV-2 RNA in the treatment process of WWTP has not been widely studied yet; therefore, in this study, we aimed to evaluate the efficiency of treatment processes in reducing SARS-CoV-2 RNA levels in wastewater. The treatment process of three WWTPs of the Parisian area in France was monitored on six different weeks over a period of 2 months (from April 14 to June 9, 2021). SARS-CoV-2 RNA copies were detected using digital polymerase chain reaction (dPCR). Investigation on the presence of variants of concern (Del69-70, E484K, and L452R) was also performed. Additionally, SARS-CoV-2 RNA loads in the WWTPs influents were expressed as the viral concentration in per population equivalent (PE) and showed a good correlation with French public health indicators (incidence rate). SARS-CoV-2 RNA loads were notably reduced along the water treatment lines of the three WWTPs studied (2.5-3.4 log reduction). Finally, very low SARS-CoV-2 RNA loads were detected in effluents (non-detected in over half of the samples) which indicated that the potential risk of the release of wastewater effluents to the environment is probably insignificant, in the case of WWTPs enabling an efficient biological removal of nitrogen.

Fate of SARS-CoV-2 coronavirus in wastewater treatment sludge during storage and thermophilic anaerobic digestion.

Since the COVID-19 outbreak has started in late 2019, SARS-CoV-2 has been widely detected in human stools and in urban wastewater. No infectious SARS-CoV-2 particles have been detected in raw wastewater until now, but it has been reported occasionally in human stools. This has raised questions on the fate of SARS-CoV-2 during wastewater treatment and notably in its end-product, wastewater treatment sludge, which is classically valorized by land spreading for agricultural amendment. In the present work, we focused on SARS-CoV-2 stability in wastewater treatment sludge, either during storage (4 °C, room temperature) or thermophilic anaerobic digestion (50 °C). Anaerobic digestion is one of the possible processes for sludge valorization. Experiments were conducted in laboratory pilots; SARS-CoV-2 detection was based on RT-quantitative PCR or RT-digital droplet PCR. In addition to SARS-CoV-2, Bovine Coronavirus (BCoV) particles were used as surrogate virus. The RNA from SARS-CoV-2 particles, inactivated or not, was close to the detection limit but stable in wastewater treatment sludge, over the whole duration of the assays at 4 °C (55 days) and at ambient temperature (∼20 °C, 25 days). By contrast, the RNA levels of BCoV and inactivated SARS-CoV-2 particles decreased rapidly during the thermophilic anaerobic digestion of wastewater treatment sludge lasting for 5 days, with final levels that were close to the detection limit. Although the particles' infectivity was not assessed, these results suggest that thermophilic anaerobic digestion is a suitable process for sludge sanitation, consistent with previous knowledge on other coronaviruses.

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.

Bacterial Consumption of T4 Phages.

The bacterial consumption of viruses not been reported on as of yet even though bacteria feed on almost anything. Viruses are widely distributed but have no acknowledged active biocontrol. Viral biomass undoubtedly reintegrates trophic cycles; however, the mechanisms of this phase still remain unknown. 13C-labelled T4 phages monitor the increase of the density of the bacterial DNA concomitant with the decrease of plaque forming units. We used 12C T4 phages as a control. T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole the source of carbon, nitrogen, and phosphorus. Bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as in the gut or in water.

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.

Microbial and functional characterization of an allochthonous consortium applied to hydrogen production from Citrus Peel Waste in batch reactor in optimized conditions.

Energy recovery from lignocellulosic waste has been studied as an alternative to the problem of inappropriate waste disposal. The present study aimed at characterizing the microbial community and the functional activity of reactors applied to H2 production through lignocellulosic waste fermentation in optimized conditions. The latter were identified by means of Rotational Central Composite Design (RCCD), applied to optimize allochthonous inoculum concentration (2.32-5.68 gTVS/L of granular anaerobic sludge), pH (4.32-7.68) and Citrus Peel Waste (CPW) concentration (1.55-28.45 g/L). After validation, the conditions identified for optimal H2 production were 4 gSTV/L of allochthonous inoculum, 29.8 g/L of CPW (substrate) and initial pH of 8.98. In these conditions, 48.47 mmol/L of H2 was obtained, which is 3.64 times higher than the concentration in unoptimized conditions (13.31 mmol H2/L using 15 g/L of CPW, 2 gTVS/L of allochthonous inoculum, pH 7.0). Acetogenesis was the predominant pathway, and maximal concentrations of 3,731 mg/L of butyric acid and 3,516 mg/L of acetic acid were observed. Regarding the metataxonomic profile, Clostridium genus was dramatically favored in the optimized condition (79.78%) when compared to the allochthonous inoculum (0.43%). It was possible to identify several genes related to H2 (i.e dehydrogenases) and volatile fatty acids (VFA) production and with cellulose degradation, especially some CAZymes from the classes Auxiliary Activities, Glycoside Hydrolases and Glycosyl Transferase. By means of differential gene expression it was observed that cellulose degradation and acetic acid production pathways were overabundant in samples from the optimized reactors, highlighting endo-ß-1,4-glucanase/cellulose, endo-ß-1,4-xylanase, ß-glucosidase, ß-mannosidase, cellulose ß-1,4-cellobiosidase, cellobiohydrolase, and others, as main the functions.

Exploring short k-mer profiles in cells and mobile elements from Archaea highlights the major influence of both the ecological niche and evolutionary history.

BACKGROUND: K-mer-based methods have greatly advanced in recent years, largely driven by the realization of their biological significance and by the advent of next-generation sequencing. Their speed and their independence from the annotation process are major advantages. Their utility in the study of the mobilome has recently emerged and they seem a priori adapted to the patchy gene distribution and the lack of universal marker genes of viruses and plasmids. To provide a framework for the interpretation of results from k-mer based methods applied to archaea or their mobilome, we analyzed the 5-mer DNA profiles of close to 600 archaeal cells, viruses and plasmids. Archaea is one of the three domains of life. Archaea seem enriched in extremophiles and are associated with a high diversity of viral and plasmid families, many of which are specific to this domain. We explored the dataset structure by multivariate and statistical analyses, seeking to identify the underlying factors. RESULTS: For cells, the 5-mer profiles were inconsistent with the phylogeny of archaea. At a finer taxonomic level, the influence of the taxonomy and the environmental constraints on 5-mer profiles was very strong. These two factors were interdependent to a significant extent, and the respective weights of their contributions varied according to the clade. A convergent adaptation was observed for the class Halobacteria, for which a strong 5-mer signature was identified. For mobile elements, coevolution with the host had a clear influence on their 5-mer profile. This enabled us to identify one previously known and one new case of recent host transfer based on the atypical composition of the mobile elements involved. Beyond the effect of coevolution, extrachromosomal elements strikingly retain the specific imprint of their own viral or plasmid taxonomic family in their 5-mer profile. CONCLUSION: This specific imprint confirms that the evolution of extrachromosomal elements is driven by multiple parameters and is not restricted to host adaptation. In addition, we detected only recent host transfer events, suggesting the fast evolution of short k-mer profiles. This calls for caution when using k-mers for host prediction, metagenomic binning or phylogenetic reconstruction.

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.

Impact of wet aerobic pretreatments on cellulose accessibility and bacterial communities in rape straw.

A new pretreatment method of lignocellulosic biomass was explored by using a wet aerobic process with an alkaline lignin and a mineral salt solution. This treatment significantly improved structural modification of rape straw used as substrate model in this study. Change in cellulose accessibility to cellulase of rape straw rose up to six fold within the first days of this pretreatment without generated significant modification of van Soest lignocellulose fractionation. The biological pretreatment apply to rape straw induced a high microbial activity revealed by quantitative PCR and sequencing techniques, suggesting that bacteria including Xanthomonadales and Sphingobacteriales may be involved in this lignocellulosic biomass transformation. Moreover, results of this work demonstrate that the endogenous microbial community associated with rape straw plays a key role in its alteration.

Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the "xyl-doc" cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.

New insights into the key microbial phylotypes of anaerobic sludge digesters under different operational conditions.

Analyses on bacterial, archaeal communities at family level and methane-production metabolism were conducted in thirteen full-scale and pilot-scale anaerobic sludge digesters. These digesters were operated at different conditions regarding solids concentration, sludge retention time, organic loading rate and feedstock composition, seeking to optimize digester capacity. Correlations between process parameters and identified microbial phylotypes were evaluated based on relative abundance of these phylotypes determined by Quantitative PCR and 16S rDNA amplicon sequencing. Results showed that, Total Solids concentration (TS), among the evaluated operational parameters, demonstrated the most positive correlation with chemical parameters (including NH3 and VFAs) and significant impact on the abundance of key microbial phylotypes regardless of other factors. Digesters were grouped into 'Higher-TS' with higher stress (TS > 44 g/L, NH3 > 90 mg/L, VFAs > 300 mg/L) and 'Lower-TS' under easier status (TS ≤ 44 g/L, NH3 < 120 mg/L, VFAs < 525 mg/L) in this study. We identified the key microbial phylotypes, i.e. the most abundant and discriminating populations, in 'Higher-TS' digesters with high biogas production rate, which were the class Clostridia, the family Methanosarcinaceae and the order Methanobacteriales. Thermoanaerobacteraceae and Syntrophomonadaceae were identified as key families of Clostridia. Methane was produced both from acetoclastic and hydrogenotrophic methanogenesis. By contrast, in 'Higher-TS' digesters with low biogas production rate, the classes Alpha-, Beta- and Gamma-proteobacteria were detected in higher percentages, of which Rhodobacteraceae, Comamonadaceae and Xanthomonadaceae were the most abundant families respectively, and Methanomicrobiales was the prevailing methanogen order. Consistently, hydrogenotrophic pathway was predominant for methanogenesis, indicating existence of syntrophic acetate oxidation in such 'high-stress', low biogas production rate digesters. These microbial phylotypes were therefore considered to be associated to 'Higher-TS' operation. In 'Lower-TS' digesters, the abundance of the class Delta-proteobacteria, the families Anaerolineaceae, Rikenellaceae, Candidatus Cloacamonas and Methanosaetaceae was obviously higher compared with those in 'Higher-TS' digesters, which were thus considered to be marker phylotypes of easy status. The influence of TS and NH3 on the microbiome should be considered when a 'TS-increasing' strategy is applied to increase digester capacity.

Community shifts within anaerobic digestion microbiota facing phenol inhibition: Towards early warning microbial indicators?

Performance stability is a key operational issue for anaerobic digestion (AD) and phenolic compounds are regularly mentioned as a major cause of digester failures. To get more insights into AD microbiota response to a wide range of inhibition levels, anaerobic batch toxicity assays were conducted with ten phenol concentrations up to 5.00 g/L. Final AD performance was not impaired up to 1.00 g/L. However, progressive shifts in microbial community structure were detected from 0.50 g/L. The methanogenic function was maintained along with increasing initial phenol concentrations up to 2.00 g/L thanks to the emergence of genus Methanoculleus at the expense of Methanosarcina. Within syntrophic populations, family Syntrophomonadaceae proportion was gradually reduced by phenol while Synergistaceae gained in importance in the microbiome. Moreover, at 2.00 g/L, the relative abundance of families belonging to order Clostridiales dropped, leading to the predominance of populations assigned to order Bacteroidales even though it did not prevent final AD performance deterioration. It illustrates the high level of adaptability of archaeal and bacterial communities and suggests the possibility of determining early warning microbial indicators associated with phenol inhibition.

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).

Co-inoculating ruminal content neither provides active hydrolytic microbes nor improves methanization of ¹³C-cellulose in batch digesters.

Cellulose hydrolysis often limits the kinetics and efficiency of anaerobic degradation in industrial digesters. In animal digestive systems, specialized microorganisms enable cellulose biodegradation at significantly higher rates. This study aims to assess the potential of ruminal microbial communities to settle and to express their cellulolytic properties in anaerobic digesters. Cellulose-degrading batch incubations were co-inoculated with municipal solid waste digester sludge and ruminal content. ¹³C-labeled cellulose degradation was described over time with Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry. Results were linked to the identification of the microorganisms assimilating ¹³C and to the monitoring of their relative dynamics. Cellulose degradation in co-inoculated incubations was efficient but not significantly improved. Transient disturbances in degradation pathways occurred, as revealed by propionate accumulation. Automated Ribosomal Intergenic Spacer Analysis dynamics and pyrosequencing revealed that expected classes of Bacteria and Archaea were active and degraded cellulose. However, despite the favorable co-inoculation conditions, molecular tools also revealed that no ruminal species settled in the bioreactors. Other specific parameters were probably needed for this to happen. This study shows that exploiting the rumen's cellulolytic properties in anaerobic digesters is not straightforward. Co-inoculation can only be successful if ruminal microorganisms manage to thrive in the anaerobic digester and outcompete native microorganisms, which requires specific nutritional and environmental parameters, and a meticulous reproduction of the selection pressure encountered in the rumen.

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity.

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.

[Enigmatic archaeal viruses]. / Énigmatiques virus d'archées.

Viruses infecting microorganisms of the third domain of life, Archaea, are still poorly characterized: to date, only about fifty of these viruses have been isolated. Their hosts are hyperthermophilic, acidothermophilic, and extreme halophilic or methanogenic archaea. Their morphotypes are highly diverse and their gene content is very specific. Some of these viruses have developed extraordinary mechanisms to open the cell wall thanks to the formation of exceptional pyramidal nanostructures. The still limited knowledge about the biology of archaeoviruses should develop rapidly in the coming years.

Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon.

The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S. islandicus strains isolated from Iceland, Russia and USA revealed a highly syntenic common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin-antitoxin genes and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we have developed a genetic system for S. islandicus LAL14/1 and created ΔpyrEF and ΔCRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus-host interactions and the CRISPR/Cas defence mechanism in Archaea.

The archeoviruses.

Since their discovery in the early 1980s, viruses that infect the third domain of life, the Archaea, have captivated our attention because of their virions' unusual morphologies and proteins, which lack homologues in extant databases. Moreover, the life cycles of these viruses have unusual features, as revealed by the recent discovery of a novel virus egress mechanism that involves the formation of specific pyramidal structures on the host cell surface. The available data elucidate the particular nature of the archaeal virosphere and shed light on questions concerning the origin and evolution of viruses and cells. In this review, we summarize the current knowledge of archeoviruses, their interaction with hosts and plasmids and their role in the evolution of life.

A unique virus release mechanism in the Archaea.

Little is known about the infection cycles of viruses infecting cells from Archaea, the third domain of life. Here, we demonstrate that the virions of the archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. Large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the S-layer; they eventually open out, thus creating large apertures through which virions escape the cell. We also demonstrate that massive degradation of the host chromosomes occurs because of virus infection, and that virion assembly occurs in the cytoplasm. Furthermore, intracellular viral DNA is visualized by flow cytometry. The results show that SIRV2 is a lytic virus, and that the host cell dies as a consequence of elaborated mechanisms orchestrated by the virus. The generation of specific cellular structures for a distinct step of virus life cycle is known in eukaryal virus-host systems but is unprecedented in cells from other domains.