BackgroundAnaerobic digestion (AD) is a microbe-driven process of biomass decomposition to CH4 and CO2. In addition to renewable and cost-effective energy production, AD has emerged in the European Union as an environmentally friendly model of bio-waste valorisation and nutrient recycling. Nevertheless, due to the high diversity of uncharacterised microbes, a typical AD microbiome is still considered as “dark matter”.ResultsUsing the high-throughput sequencing of small rRNA gene, and a monthly monitoring of the physicochemical parameters for 20 different mesophilic full-scale bioreactors over 1 year, we generated a detailed view of AD microbial ecology towards a better understanding of factors that influence and shape these communities. By studying the broadly distributed OTUs present in over 80% of analysed samples, we identified putatively important core bacteria and archaea to the AD process that accounted for over 70% of the whole microbial community relative abundances. AD reactors localised at the wastewater treatment plants were shown to operate with distinct core microbiomes than the agricultural and bio-waste treating biogas units. We also showed that both the core microbiomes were composed of low (with average community abundance ≤ 1%) and highly abundant microbial populations; the vast majority of which remains yet uncharacterised, e.g. abundant candidate Cloacimonetes. Using non-metric multidimensional scaling, we observed microorganisms grouping into clusters that well reflected the origin of the samples, e.g. wastewater versus agricultural and bio-waste treating biogas units. The calculated diversity patterns differed markedly between the different community clusters, mainly due to the presence of highly diverse and dynamic transient species. Core microbial communities appeared relatively stable over the monitoring period.ConclusionsIn this study, we characterised microbial communities in different AD systems that were monitored over a 1-year period. Evidences were shown to support the concept of a core community driving the AD process, whereas the vast majority of dominant microorganisms remain yet to be characterised.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1195-8) contains supplementary material, which is available to authorized users.

BackgroundVolatile fatty acid intoxication (acidosis), a common process failure recorded in anaerobic reactors, leads to drastic losses in methane production. Unfortunately, little is known about the microbial mechanisms underlining acidosis and the potential to recover the process. In this study, triplicate mesophilic anaerobic reactors of 100 L were exposed to acidosis resulting from an excessive feeding with sugar beet pulp and were compared to a steady-state reactor.ResultsStable operational conditions at the beginning of the experiment initially led to similar microbial populations in the four reactors, as revealed by 16S rRNA gene T-RFLP and high-throughput amplicon sequencing. Bacteroidetes and Firmicutes were the two dominant phyla, and although they were represented by a high number of operational taxonomic units, only a few were dominant. Once the environment became deterministic (selective pressure from an increased substrate feeding), microbial populations started to diverge between the overfed reactors. Interestingly, most of bacteria and archaea showed redundant functional adaptation to the changing environmental conditions. However, the dominant Bacteroidales were resistant to high volatile fatty acids content and low pH. The severe acidosis did not eradicate archaea and a clear shift in archaeal populations from acetotrophic to hydrogenotrophic methanogenesis occurred in the overfed reactors. After 11 days of severe acidosis (pH 5.2 ± 0.4), the process was quickly recovered (restoration of the biogas production with methane content above 50 %) in the overfed reactors, by adjusting the pH to around 7 using NaOH and NaHCO3.ConclusionsIn this study we show that once the replicate reactors are confronted with sub-optimal conditions, their microbial populations start to evolve differentially. Furthermore the alterations of commonly used microbial parameters to monitor the process, such as richness, evenness and diversity indices were unsuccessful to predict the process failure. At the same time, we tentatively propose the replacement of the dominant Methanosaeta sp. in this case by Methanoculleus sp., to be a potential warning indicator of acidosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0309-9) contains supplementary material, which is available to authorized users.

Miscanthus sp. biomass could satisfy future biorefinery value chains. However, its use is largely untapped due to high recalcitrance. The termite and its gut microbiome are considered the most efficient lignocellulose degrading system in nature. Here, we investigate at holobiont level the dynamic adaptation of Cortaritermes sp. to imposed Miscanthus diet, with a long-term objective of overcoming lignocellulose recalcitrance. We use an integrative omics approach combined with enzymatic characterisation of carbohydrate active enzymes from termite gut Fibrobacteres and Spirochaetae. Modified gene expression profiles of gut bacteria suggest a shift towards utilisation of cellulose and arabinoxylan, two main components of Miscanthus lignocellulose. Low identity of reconstructed microbial genomes to closely related species supports the hypothesis of a strong phylogenetic relationship between host and its gut microbiome. This study provides a framework for better understanding the complex lignocellulose degradation by the higher termite gut system and paves a road towards its future bioprospecting.

BackgroundThanks to specific adaptations developed over millions of years, the efficiency of lignin, cellulose and hemicellulose decomposition of higher termite symbiotic system exceeds that of many other lignocellulose utilizing environments. Especially, the examination of its symbiotic microbes should reveal interesting carbohydrate-active enzymes, which are of primary interest for the industry. Previous metatranscriptomic reports (high-throughput mRNA sequencing) highlight the high representation and overexpression of cellulose and hemicelluloses degrading genes in the termite hindgut digestomes, indicating the potential of this technology in search for new enzymes. Nevertheless, several factors associated with the material sampling and library preparation steps make the metatranscriptomic studies of termite gut prokaryotic symbionts challenging.MethodsIn this study, we first examined the influence of the sampling strategy, including the whole termite gut and luminal fluid, on the diversity and the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. Secondly, we evaluated different commercially available kits combined in two library preparative pipelines for the best bacterial mRNA enrichment strategy.ResultsWe showed that the sampling strategy did not significantly impact the generated results, both in terms of the representation of the microbes and their transcriptomic profiles. Nevertheless collecting luminal fluid reduces the co-amplification of unwanted RNA species of host origin. Furthermore, for the four studied higher termite species, the library preparative pipeline employing Ribo-Zero Gold rRNA Removal Kit “Epidemiology” in combination with Poly(A) Purist MAG kit resulted in a more efficient rRNA and poly-A-mRNAdepletion (up to 98.44% rRNA removed) than the pipeline utilizing MICROBExpress and MICROBEnrich kits. High correlation of both Ribo-Zero and MICROBExpresse depleted gene expression profiles with total non-depleted RNA-seq data has been shown for all studied samples, indicating no systematic skewing of the studied pipelines.ConclusionsWe have extensively evaluated the impact of the sampling strategy and library preparation steps on the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. The presented methodological approach has great potential to enhance metatranscriptomic studies of the higher termite intestinal flora and to unravel novel carbohydrate-active enzymes.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4076-9) contains supplementary material, which is available to authorized users.

The goal of this study was to investigate how the microbial community structure establishes during the start-up phase of a full-scale farm anaerobic reactor inoculated with stale and cold cattle slurry. The 16S/18S high-throughput amplicon sequencing results showed an increase of the bacterial, archaeal and eukaryotic diversity, evenness and richness during the settlement of the mesophilic anaerobic conditions. When a steady performing digestion process was reached, the microbial diversity, evenness and richness decreased, indicating the establishment of a few dominant microbial populations, best adapted to biogas production. Interestingly, among the environmental parameters, the temperature, alkalinity, free-NH3, total solids and O2 content were found to be the main drivers of microbial dynamics. Interactions between eukaryotes, characterized by a high number of unknown organisms, and the bacterial and archaeal communities were also evidenced, suggesting that eukaryotes might play important roles in the anaerobic digestion process.

SummaryAlthough viruses are not the key players of the anaerobic digestion process, they may affect the dynamics of bacterial and archaeal populations involved in biogas production. Until now viruses have received very little attention in this specific habitat; therefore, as a first step towards their characterization, we optimized a virus filtration protocol from anaerobic sludge. Afterwards, to assess dsDNA and RNA viral diversity in sludge samples from nine different reactors fed either with waste water, agricultural residues or solid municipal waste plus agro-food residues, we performed metagenomic analyses. As a result we showed that, while the dsDNA viromes (21 assigned families in total) were dominated by dsDNA phages of the order Caudovirales, RNA viruses (14 assigned families in total) were less diverse and were for the main part plant-infecting viruses. Interestingly, less than 2% of annotated contigs were assigned as putative human and animal pathogens. Our study greatly extends the existing view of viral genetic diversity in methanogenic reactors and shows that these viral assemblages are distinct not only among the reactor types but also from nearly 30 other environments already studied, including the human gut, fermented food, deep sea sediments and other aquatic habitats.

Increased hydrolysis of easily digestible biomass may lead to acidosis of anaerobic reactors and decreased methane production. Previously, it was shown that the structure of microbial communities changed during acidosis; however, once the conditions are back to optimal, biogas (initially CO2) production quickly restarts. This suggests the retention of the community functional redundancy during the process failure. In this study, with the use of metagenomics and downstream bioinformatics analyses, we characterize the carbohydrate hydrolytic potential of the microbial community, with a special focus on acidosis. To that purpose, carbohydrate-active enzymes were identified, and to further link the community hydrolytic potential with key microbes, bacterial genomes were reconstructed. In addition, we characterized biochemically the specificity and activity of selected enzymes, thus verifying the accuracy of the in silico predictions. The results confirm the retention of the community hydrolytic potential during acidosis and indicate Bacteroidetes to be largely involved in biomass degradation. Bacteroidetes showed higher diversity and genomic content of carbohydrate hydrolytic enzymes that might favor the dominance of this phylum over other bacteria in some anaerobic reactors. The combination of bioinformatic analyses and activity tests enabled us to propose a model of acetylated glucomannan degradation by Bacteroidetes. IMPORTANCE The enzymatic hydrolysis of lignocellulosic biomass is mainly driven by the action of carbohydrate-active enzymes. By characterizing the gene profiles at the different stages of the anaerobic digestion experiment, we showed that the microbiome retains its hydrolytic functional redundancy even during severe acidosis, despite significant changes in taxonomic composition. By analyzing reconstructed bacterial genomes, we demonstrate that Bacteroidetes hydrolytic gene diversity likely favors the abundance of this phylum in some anaerobic digestion systems. Further, we observe genetic redundancy within the Bacteroidetes group, which accounts for the preserved hydrolytic potential during acidosis. This work also uncovers new polysaccharide utilization loci involved in the deconstruction of various biomasses and proposes the model of acetylated glucomannan degradation by Bacteroidetes. Acetylated glucomannan-enriched biomass is a common substrate for many industries, including pulp and paper production. Using naturally evolved cocktails of enzymes for biomass pretreatment could be an interesting alternative to the commonly used chemical pretreatments.

Background: Termites are among the most successful insect lineages on the globe and are responsible for providing numerous ecosystem services. They mainly feed on wood and other plant material at different stages of humification. Lignocellulose is often a principal component of such plant diet, and termites largely rely on their symbiotic microbiota and associated enzymes to decompose their food efficiently. While lower termites and their gut flagellates were given larger scientific attention in the past, the gut lignocellulolytic bacteria of higher termites remain less explored. Therefore, in this study, we investigated the structure and function of gut prokaryotic microbiomes from 11 higher termite genera representative of Syntermitinae, Apicotermitinae, Termitidae and Nasutitermitinae subfamilies, broadly grouped into plant fibre-and soil-feeding termite categories. Results: Despite the different compositional structures of the studied termite gut microbiomes, reflecting well the diet and host lineage, we observed a surprisingly high functional congruency between gut metatranscriptomes from both feeding groups. The abundance of transcripts encoding for carbohydrate active enzymes as well as expression and diversity profiles of assigned glycoside hydrolase families were also similar between plant fibre-and soil-feeding termites. Yet, dietary imprints highlighted subtle metabolic differences specific to each feeding category. Roughly, 0.18% of de novo reconstructed gene transcripts were shared between the different termite gut microbiomes, making each termite gut a unique reservoir of genes encoding for potentially industrially applicable enzymes, e.g. relevant to biomass degradation. Taken together, we demonstrated the functional equivalence in microbial populations across different termite hosts. Conclusions: Our results provide valuable insight into the bacterial component of the termite gut system and significantly expand the inventory of termite prokaryotic genes participating in the deconstruction of plant biomass.