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Microbiology is founded on well-known model organisms. For example, the majority of our fundamental knowledge regarding the quantitative levels of DNA, RNA, and protein backdates to keystone pure culture-based studies. Nowadays, meta-omic approaches allow us to directly access the molecules that constitute microbes and microbial communities, however due to a lack of absolute measurements, many original culture-derived “microbiology statutes” have not been updated or adapted to more complex microbiome settings. Within a cellulose-degrading and methanogenic consortium, we temporally measured genome-centric absolute RNA and protein levels per gene, and obtained a protein-to-RNA ratio of 102-104 for bacterial populations, whereas Archaeal RNA/protein dynamics (103-105: Methanothermobacter thermoautotrophicus) were more comparable to Eukaryotic representatives humans and yeast. The linearity between transcriptome and proteome had a population-specific change over time, highlighting a minimal subset of four functional carriers (cellulose degrader, fermenter, syntrophic acetate-oxidizer and methanogen) that coordinated their respective metabolisms, cumulating in the overarching community phenotype of converting polysaccharides to methane. Our findings show that upgrading multi-omic toolkits with traditional absolute measurements unlocks the scaling of core biological questions to dynamic and complex microbiomes, creating a deeper insight into inter-organismal relationships that drive the greater community function.

Beneficial modulation of the gut microbiome has high-impact implications not only in humans, but also in livestock that sustain our current societal needs. In this context, we have tailored an acetylated galactoglucomannan (AcGGM) fibre to match unique enzymatic capabilities of Roseburia and Faecalibacterium species, both renowned butyrate-producing gut commensals. The accuracy of AcGGM was tested within the complex endogenous gut microbiome of pigs, wherein we resolved 355 metagenome-assembled genomes together with quantitative metaproteomes. In AcGGM-fed pigs, both target populations differentially expressed AcGGM-specific polysaccharide utilization loci, including novel, mannan-specific esterases that are critical to its deconstruction. However, AcGGM-inclusion also manifested a “butterfly effect”, whereby numerous metabolic changes and interdependent cross-feeding pathways were detected in neighboring non-mannolytic populations that produce short-chain fatty acids. Our findings show that intricate structural features and acetylation patterns of dietary fibre can be customized to specific bacterial populations, with potential to create greater modulatory effects at large.

Norway is the largest producer and exporter of farmed Atlantic salmon (Salmo salar) worldwide. Skin disorders correlated with bacterial infections represent an important challenge for fish farmers due to the economic losses caused. Little is known about this topic, thus studying the skin&ndash, mucus of Salmo salar and its bacterial community depict a step forward in understanding fish welfare in aquaculture. In this study, we used label free quantitative mass spectrometry to investigate the skin&ndash, mucus proteins associated with both Atlantic salmon and bacteria. In particular, the microbial temporal proteome dynamics during nine days of mucus incubation with sterilized seawater was investigated, in order to evaluate their capacity to utilize mucus components for growth in this environment. At the start of the incubation period, the largest proportion of proteins (~99%) belonged to the salmon and many of these proteins were assigned to protecting functions, confirming the defensive role of mucus. On the contrary, after nine days of incubation, most of the proteins detected were assigned to bacteria, mainly to the genera Vibrio and Pseudoalteromonas. Most of the predicted secreted proteins were affiliated with transport and metabolic processes. In particular, a large abundance and variety of bacterial proteases were observed, highlighting the capacity of bacteria to degrade the skin&ndash, mucus proteins of Atlantic salmon.

Ruminants have co-evolved with symbiotic rumen microbiota, which readily convert ingested plant fibres into the nutrients they need to sustain their growth and maintenance. Fibre degradation within the rumen microbiome has been attributed to a limited number of cultivable representatives, which has restricted our ability to understand the different enzymatic machineries that exist. However, via a combination of culturing, meta-omics, bioinformatics, biochemistry and enzymology, we are beginning to expand our insight into the different fibre-digesting strategies that rumen microbiota employ. We discuss findings from studies on well-known Ruminococcus, Fibrobacter and Prevotella isolates, as well as those from poorly understood and as-yet uncultured Bacteroidetes lineages. Collectively, these approaches have revealed new mechanistic information related to the hydrolytic capacity of cellulosomes, free enzymes, outer membrane vesicles, polysaccharide utilization loci and large multi-modular enzymes, which are generating deeper insights into the intricate microbial networks that engage in ruminal fibre digestion.

Norway is the largest producer and exporter of farmed Atlantic salmon (Salmo salar) worldwide. Skin disorders correlated with bacterial infections represent an important challenge for fish farmers due to the economic losses caused. Little is known about this topic, thus studying the skin–mucus of Salmo salar and its bacterial community depict a step forward in understanding fish welfare in aquaculture. In this study, we used label free quantitative mass spectrometry to investigate the skin–mucus proteins associated with both Atlantic salmon and bacteria. In particular, the microbial temporal proteome dynamics during nine days of mucus incubation with sterilized seawater was investigated, in order to evaluate their capacity to utilize mucus components for growth in this environment. At the start of the incubation period, the largest proportion of proteins (~99%) belonged to the salmon and many of these proteins were assigned to protecting functions, confirming the defensive role of mucus. On the contrary, after nine days of incubation, most of the proteins detected were assigned to bacteria, mainly to the genera Vibrio and Pseudoalteromonas. Most of the predicted secreted proteins were affiliated with transport and metabolic processes. In particular, a large abundance and variety of bacterial proteases were observed, highlighting the capacity of bacteria to degrade the skin–mucus proteins of Atlantic salmon.

The architecture of the gut bacterial ecosystem has a profound effect on the physiology and well-being of the host. Modulation of the gut microbiota and the intestinal microenvironment via administration of prebiotics represents a valuable strategy to promote host health. This work provides insights into the ability of two novel wood-derived preparations, AcGGM and AcAGX, to influence human gut microbiota composition and activity. These compounds were selectively fermented by commensal bacteria such as Bifidobacterium, Bacteroides-Prevotella, F. prausnitzii, and clostridial cluster IX spp. This promoted the microbial synthesis of acetate, propionate, and butyrate, which are beneficial to the microbial ecosystem and host colonic epithelial cells. Thus, our results demonstrate potential prebiotic properties for both AcGGM and AcAGX from lignocellulosic feedstocks. These findings represent pivotal requirements for rationally designing intervention strategies based on the dietary supplementation of AcGGM and AcAGX to improve or restore gut health., Woody biomass is a sustainable and virtually unlimited source of hemicellulosic polysaccharides. The predominant hemicelluloses in softwood and hardwood are galactoglucomannan (GGM) and arabinoglucuronoxylan (AGX), respectively. Based on the structure similarity with common dietary fibers, GGM and AGX may be postulated to have prebiotic properties, conferring a health benefit on the host through specific modulation of the gut microbiota. In this study, we evaluated the prebiotic potential of acetylated GGM (AcGGM) and highly acetylated AGX (AcAGX) obtained from Norwegian lignocellulosic feedstocks in vitro. In pure culture, both substrates selectively promoted the growth of Bifidobacterium, Lactobacillus, and Bacteroides species in a manner consistent with the presence of genetic loci for the utilization of β-manno-oligosaccharides/β-mannans and xylo-oligosaccharides/xylans. The prebiotic potential of AcGGM and AcAGX was further assessed in a pH-controlled batch culture fermentation system inoculated with healthy adult human feces. Results were compared with those obtained with a commercial fructo-oligosaccharide (FOS) mixture. Similarly to FOS, both substrates significantly increased (P

International audience; Microbial communities that degrade lignocellulosic biomass are typified by high levels of species-and strain-level complexity, as well as synergistic interactions between both cellulolytic and non-cellulolytic microorganisms. Coprothermobacter proteolyticus frequently dominates thermophilic, lignocellulose-degrading communities with wide geographical distribution, which is in contrast to reports that it ferments proteinaceous substrates and is incapable of polysaccharide hydrolysis. Here we deconvolute a highly efficient cellulose-degrading consortium (SEM1b) that is co-dominated by Clostridium (Ruminiclostridium) thermocellum and multiple heterogenic strains affiliated to C. proteolyticus. Metagenomic analysis of SEM1b recovered metagenome-assembled genomes (MAGs) for each constituent population, whereas in parallel two novel strains of C. proteolyticus were successfully isolated and sequenced. Annotation of all C. proteolyticus genotypes (two strains and one MAG) revealed their genetic acquisition of carbohydrate-active enzymes (CAZymes), presumably derived from horizontal gene transfer (HGT) events involving polysaccharide-degrading Firmicutes or Thermotogae-affiliated populations that are historically co-located. HGT material included a saccharolytic operon, from which a CAZyme was biochemically characterized and demonstrated hydrolysis of multiple hemicellulose polysaccharides. Finally, temporal genome-resolved metatranscriptomic analysis of SEM1b revealed expression of C. proteolyticus CAZymes at different SEM1b life stages as well as co-expression of CAZymes from multiple SEM1b populations, inferring deeper microbial interactions that are dedicated toward community degradation of cellulose and hemicellulose. We show that C. proteolyticus, a ubiquitous population, consists of closely related strains that have adapted via HGT to presumably degrade both oligo-and longer polysaccharides present in decaying plants and microbial cell walls, thus explaining its dominance in thermophilic anaerobic digesters on a global scale.

Cellulose is the most abundant renewable polymer on earth, but its recalcitrance limits highly efficient conversion methods for energy-related and material applications. Though microbial cellulose conversion has been studied for decades, recent advances showcased that large knowledge gaps still exist. Bacteria of the phylum Bacteroidetes are regarded as highly efficient carbohydrate metabolizers, but most species are limited to (semi)soluble glycans. A few species, including the soil bacteria C. hutchinsonii and S. myxococcoides, are regarded as cellulose specialists, but their cellulolytic mechanisms are not understood, as they do not conform to the current models for enzymatic cellulose turnover. By unraveling the proteome setups of these two bacteria during growth on both crystalline cellulose and pectin, we have taken a significant step forward in understanding their idiosyncratic mode of cellulose conversion. This report provides a plethora of new enzyme targets for improved biomass conversion., Bacteria of the phylum Bacteroidetes are regarded as highly efficient carbohydrate metabolizers, but most species are limited to (semi)soluble glycans. The soil Bacteroidetes species Cytophaga hutchinsonii and Sporocytophaga myxococcoides have long been known as efficient cellulose metabolizers, but neither species conforms to known cellulolytic mechanisms. Both species require contact with their substrate but do not encode cellulosomal systems of cell surface-attached enzyme complexes or the polysaccharide utilization loci found in many other Bacteroidetes species. Here, we have fractionated the cellular compartments of each species from cultures growing on crystalline cellulose and pectin, respectively, and analyzed them using label-free quantitative proteomics as well as enzymatic activity assays. The combined results enabled us to highlight enzymes likely to be important for cellulose conversion and to infer their cellular localization. The combined proteomes represent a wide array of putative cellulolytic enzymes and indicate specific and yet highly redundant mechanisms for cellulose degradation. Of the putative endoglucanases, especially enzymes of hitherto-unstudied glycoside hydrolase family, 8 were abundant, indicating an overlooked important role during cellulose metabolism. Furthermore, both species generated a large number of abundant hypothetical proteins during cellulose conversion, providing a treasure trove of targets for future enzymology studies. IMPORTANCE Cellulose is the most abundant renewable polymer on earth, but its recalcitrance limits highly efficient conversion methods for energy-related and material applications. Though microbial cellulose conversion has been studied for decades, recent advances showcased that large knowledge gaps still exist. Bacteria of the phylum Bacteroidetes are regarded as highly efficient carbohydrate metabolizers, but most species are limited to (semi)soluble glycans. A few species, including the soil bacteria C. hutchinsonii and S. myxococcoides, are regarded as cellulose specialists, but their cellulolytic mechanisms are not understood, as they do not conform to the current models for enzymatic cellulose turnover. By unraveling the proteome setups of these two bacteria during growth on both crystalline cellulose and pectin, we have taken a significant step forward in understanding their idiosyncratic mode of cellulose conversion. This report provides a plethora of new enzyme targets for improved biomass conversion.

Background In nature, obligate herbivorous ruminants have a close symbiotic relationship with their gastrointestinal microbiome, which proficiently deconstructs plant biomass. Despite decades of research, lignocellulose degradation in the rumen has thus far been attributed to a limited number of culturable microorganisms. Here, we combine meta-omics and enzymology to identify and describe a novel Bacteroidetes family (“Candidatus MH11”) composed entirely of uncultivated strains that are predominant in ruminants and only distantly related to previously characterized taxa. Results The first metabolic reconstruction of Ca. MH11-affiliated genome bins, with a particular focus on the provisionally named “Candidatus Paraporphyromonas polyenzymogenes”, illustrated their capacity to degrade various lignocellulosic substrates via comprehensive inventories of singular and multi-modular carbohydrate active enzymes (CAZymes). Closer examination revealed an absence of archetypical polysaccharide utilization loci found in human gut microbiota. Instead, we identified many multi-modular CAZymes putatively secreted via the Bacteroidetes-specific type IX secretion system (T9SS). This included cellulases with two or more catalytic domains, which are modular arrangements that are unique to Bacteroidetes species studied to date. Core metabolic proteins from Ca. P. polyenzymogenes were detected in metaproteomic data and were enriched in rumen-incubated plant biomass, indicating that active saccharification and fermentation of complex carbohydrates could be assigned to members of this novel family. Biochemical analysis of selected Ca. P. polyenzymogenes CAZymes further iterated the cellulolytic activity of this hitherto uncultured bacterium towards linear polymers, such as amorphous and crystalline cellulose as well as mixed linkage β-glucans. Conclusion We propose that Ca. P. polyenzymogene genotypes and other Ca. MH11 members actively degrade plant biomass in the rumen of cows, sheep and most likely other ruminants, utilizing singular and multi-domain catalytic CAZymes secreted through the T9SS. The discovery of a prominent role of multi-modular cellulases in the Gram-negative Bacteroidetes, together with similar findings for Gram-positive cellulosomal bacteria (Ruminococcus flavefaciens) and anaerobic fungi (Orpinomyces sp.), suggests that complex enzymes are essential and have evolved within all major cellulolytic dominions inherent to the rumen. Electronic supplementary material The online version of this article (10.1186/s40168-018-0421-8) contains supplementary material, which is available to authorized users.

Responses of bacterial and archaeal communities to the addition of straw during anaerobic digestion of manure at different temperatures (37°C, 44°C and 52°C) were investigated using five laboratory-scale semi-continuous stirred tank reactors. The results revealed that including straw as co-substrate decreased the species richness for bacteria, whereas increasing the operating temperature decreased the species richness for both archaea and bacteria, and also the evenness of the bacteria. Taxonomic classifications of the archaeal community showed that Methanobrevibacter dominated in the manure samples, while Methanosarcina dominated in all digesters regardless of substrate. Increase of the operating temperature to 52°C led to increased relative abundance of Methanoculleus and Methanobacterium. Among the bacteria, the phyla Firmicutes and Bacteroidetes dominated within all samples. Compared with manure itself, digestion of manure resulted in a higher abundance of an uncultured class WWE1 and lower abundance of Bacilli. Adding straw to the digesters increased the level of Bacteroidia, while increasing the operating temperature decreased the level of this class and instead increased the relative abundance of an uncultured genus affiliated to order MBA08 (Clostridia). A considerable fraction of bacterial sequences could not be allocated to genus level, indicating that novel phylotypes are resident in these communities.

A new biogas process is initiated by adding a microbial community, typically in the form of a sample collected from a functional biogas plant. This inoculum has considerable impact on the initial performance of a biogas reactor, affecting parameters such as stability, biogas production yields and the overall efficiency of the anaerobic digestion process. In this study, we have analyzed changes in the microbial composition and performance of an inoculum during storage using barcoded pyrosequencing of bacterial and archaeal 16S ribosomal RNA (rRNA) genes, and determination of the biomethane potential, respectively. The inoculum was stored at room temperature, 4 and -20 °C for up to 11 months and cellulose was used as a standard substrate to test the biomethane potential. Storage up to 1 month resulted in similar final methane yields, but the rate of methane production was reduced by storage at -20 °C. Longer storage times resulted in reduced methane yields and slower production kinetics for all storage conditions, with room temperature and frozen samples consistently giving the best and worst performance, respectively. Both storage time and temperature affected the microbial community composition and methanogenic activity. In particular, fluctuations in the relative abundance of Bacteroidetes were observed. Interestingly, a shift from hydrogenotrophic methanogens to methanogens with the capacity to perform acetoclastic methanogensis was observed upon prolonged storage. In conclusion, this study suggests that biogas inocula may be stored up to 1 month with low loss of methanogenic activity, and identifies bacterial and archaeal species that are affected by the storage.

Gut microbiota associations through habitat transitions are fundamentally important yet poorly understood. One such habitat transition is the migration from freshwater to saltwater for anadromous fish, such as salmon. The aim of the current work was therefore to determine the freshwater-to-saltwater transition impact on the gut microbiota in farmed Atlantic salmon, with dietary interventions resembling freshwater and saltwater diets with respect to fatty acid composition. Using deep 16S rRNA gene sequencing and quantitative PCR, we found that the freshwater-to-saltwater transition had a major association with the microbiota composition and quantity, while diet did not show significant associations with the microbiota. In saltwater there was a 100-fold increase in bacterial quantity, with a relative increase of Firmicutes and a relative decrease of both Actinobacteria and Proteobacteria . Irrespective of an overall shift in microbiota composition from freshwater to saltwater, we identified three core clostridia and one Lactobacillus -affiliated phylotype with wide geographic distribution that were highly prevalent and co-occurring. Taken together, our results support the importance of the dominating bacteria in the salmon gut, with the freshwater microbiota being immature. Due to the low number of potentially host-associated bacterial species in the salmon gut, we believe that farmed salmon can represent an important model for future understanding of host-bacterium interactions in aquatic environments. IMPORTANCE Little is known about factors affecting the interindividual distribution of gut bacteria in aquatic environments. We have shown that there is a core of four highly prevalent and co-occurring bacteria irrespective of feed and freshwater-to-saltwater transition. The potential host interactions of the core bacteria, however, need to be elucidated further.

Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

Microorganisms play a primary role in regulating biogeochemical cycles and are a valuable source of enzymes that have biotechnological applications, such as carbohydrate-active enzymes (CAZymes). However, the inability to culture the majority of microorganisms that exist in natural ecosystems using common culture-dependent techniques restricts access to potentially novel cellulolytic bacteria and beneficial enzymes. The development of molecular-based culture-independent methods such as metagenomics enables researchers to study microbial communities directly from environmental samples, and presents a platform from which enzymes of interest can be sourced. We outline key methodological stages that are required as well as describe specific protocols that are currently used for metagenomic projects dedicated to CAZyme discovery.

Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (β-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.

This is a post-peer-review, pre-copyedit version of an article published in Microbial Ecology. The final authenticated version is available online at: https://doi.org/10.1007/s00248-013-0279-z. Highly cellulolytic bacterial species such as Ruminococcus flavefaciens are regarded essential for the microbial breakdown of cellulose in the rumen. We have investigated the effect of ruminal dosing of R. flavefaciens strain 8/94-32 during realimentation of starved reindeer (males, n = 3). Microbiome function measured as in situ digestion of cellulose and food pellets (percent DMD; dry matter disappearance) decreased after probiotic dosing. Microbial community analyses (>100,000 16S rDNA gene sequences for 27 samples) demonstrated that ruminal dosing influenced the microbiome structure; reflected by increased phylogenetic distances from background samples (unweighted UniFrac analysis) and reduced species diversity and evenness. Despite the inability to detect strain 8/94-32 post-dosing, the relative abundance of its affiliate family Ruminococcaceae remained consistent throughout the trial, whilst a dominant peak in the genus Prevotella and decline in uncharacterized Bacteroidetes (uBacNR) were observed in treatment samples. No clear relationships were observed between the relative abundance of Ruminococcaceae, Prevotella and uBacNR with cellulose DMD; however, Prevotella (negative) and uBacNR (positive) exhibited relationships with pellet DMD. These unexpected effects of ruminal dosing of a cellulolytic bacterium on digestibility are relevant for other studies on rumen manipulation.

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.

Microorganisms play a primary role in regulating biogeochemical cycles and are a valuable source of enzymes that have biotechnological applications, such as carbohydrate-active enzymes (CAZymes). However, the inability to culture the majority of microorganisms that exist in natural ecosystems using common culture-dependent techniques restricts access to potentially novel cellulolytic bacteria and beneficial enzymes. The development of molecular-based culture-independent methods such as metagenomics enables researchers to study microbial communities directly from environmental samples, and presents a platform from which enzymes of interest can be sourced. We outline key methodological stages that are required as well as describe specific protocols that are currently used for metagenomic projects dedicated to CAZyme discovery.

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH 3 -N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter- affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum “ Atribacteria .” These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and β-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor. IMPORTANCE Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the β-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane.

Background Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown. Results Here we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm. Conclusions By combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0674-z) contains supplementary material, which is available to authorized users.

Publisher's version, source http://doi.org/10.1099/mgen.0.000066 The faecal microbiota of muskoxen (n=3) pasturing on Ryøya (69° 33′ N 18° 43′ E), Norway, in late September was characterized using high-throughput sequencing of partial 16S rRNA gene regions. A total of 16 209 high-quality sequence reads from bacterial domains and 19 462 from archaea were generated. Preliminary taxonomic classifications of 806 bacterial operational taxonomic units (OTUs) resulted in 53.7–59.3 % of the total sequences being without designations beyond the family level. Firmicutes (70.7–81.1 % of the total sequences) and Bacteroidetes (16.8–25.3 %) constituted the two major bacterial phyla, with uncharacterized members within the family Ruminococcaceae (28.9–40.9 %) as the major phylotype. Multiple-library comparisons between muskoxen and other ruminants indicated a higher similarity for muskoxen faeces and reindeer caecum (P>0.05) and some samples from cattle faeces. The archaeal sequences clustered into 37 OTUs, with dominating phylotypes affiliated to the methane-producing genus Methanobrevibacter (80–92 % of the total sequences). UniFrac analysis demonstrated heterogeneity between muskoxen archaeal libraries and those from reindeer and roe deer (P=1.0e-02, Bonferroni corrected), but not with foregut fermenters. The high proportion of cellulose-degrading Ruminococcus-affiliated bacteria agrees with the ingestion of a highly fibrous diet. Further experiments are required to elucidate the role played by these novel bacteria in the digestion of this fibrous Artic diet eaten by muskoxen.

DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1 kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.

Publisher's version, source: http://dx.doi.org/10.1371/journal.pone.0155213 . Reindeer (Rangifer tarandus tarandus) are large Holarctic herbivores whose heterogeneous diet has led to the development of a unique gastrointestinal microbiota, essential for the digestion of arctic flora, which may include a large proportion of lichens during winter. Lichens are rich in plant secondary metabolites, which may affect members of the gut microbial consortium, such as the methane-producing methanogenic archaea. Little is known about the effect of lichen consumption on the rumen and cecum microbiotas and how this may affect methanogenesis in reindeer. Here, we examined the effects of dietary lichens on the reindeer gut microbiota, especially methanogens. Samples from the rumen and cecum were collected from two groups of reindeer, fed either lichens (Ld: n = 4), or a standard pelleted feed (Pd: n = 3). Microbial densities (methanogens, bacteria and protozoa) were quantified using quantitative real-time PCR and methanogen and bacterial diversities were determined by 454 pyrosequencing of the 16S rRNA genes. In general, the density of methanogens were not significantly affected (p>0.05) by the intake of lichens. Methanobrevibacter constituted the main archaeal genus (>95% of reads), with Mbr. thaueri CW as the dominant species in both groups of reindeer. Bacteria belonging to the uncharacterized Ruminococcaceae and the genus Prevotella were the dominant phylotypes in the rumen and cecum, in both diets (ranging between 16–38% total sequences). Bacteria belonging to the genus Ruminococcus (3.5% to 0.6%; p = 0.001) and uncharacterized phylotypes within the order Bacteroidales (8.4% to 1.3%; p = 0.027), were significantly decreased in the rumen of lichen-fed reindeer, but not in the cecum (p = 0.2 and p = 0.087, respectively). UniFrac-based analyses showed archaeal and bacterial libraries were significantly different between diets, in both the cecum and the rumen (vegan::Adonis: pseudo-F

We describe here the role of muramidases present in clones of metagenomic DNA that result in cell aggregation and biofilm formation by Escherichia coli. The metagenomic clones were obtained from uncultured Lachnospiraceae-affiliated bacteria resident in the foregut microbiome of the Tammar wallaby. One of these fosmid clones (p49C2) was chosen for more detailed studies and a variety of genetic methods were used to delimit the region responsible for the phenotype to an open reading frame of 1425 bp. Comparative sequence analysis with other fosmid clones giving rise to the same phenotype revealed the presence of muramidase homologues with the same modular composition. Phylogenetic analysis of the fosmid sequence data assigned these fosmid inserts to recently identified, but uncultured, phylogroups of Lachnospiraceae believed to be numerically dominant in the foregut microbiome of the Tammar wallaby. The muramidase is a modular protein containing putative N-acetylmuramoyl-L-alanine amidase and an endo-β-N-acetylglucosaminidase catalytic module, with a similar organization and functional properties to some Staphylococcal autolysins that also confer adhesive properties and biofilm formation. We also show here that the cloned muramidases result in the production of extracellular DNA, which appears to be the key for biofilm formation and autoaggregation. Collectively, these findings suggest that biofilm formation and cell aggregation in gut microbiomes might occur via the concerted action of carbohydrate-active enzymes and the production of extracellular DNA to serve as a biofilm scaffold.

Metagenomic and bioinformatic approaches were used to characterize plant biomass conversion within the foregut microbiome of Australia's “model” marsupial, the Tammar wallaby ( Macropus eugenii ). Like the termite hindgut and bovine rumen, key enzymes and modular structures characteristic of the “free enzyme” and “cellulosome” paradigms of cellulose solubilization remain either poorly represented or elusive to capture by shotgun sequencing methods. Instead, multigene polysaccharide utilization loci-like systems coupled with genes encoding β-1,4-endoglucanases and β-1,4-endoxylanases—which have not been previously encountered in metagenomic datasets—were identified, as were a diverse set of glycoside hydrolases targeting noncellulosic polysaccharides. Furthermore, both rrs gene and other phylogenetic analyses confirmed that unique clades of the Lachnospiraceae, Bacteroidales, and Gammaproteobacteria are predominant in the Tammar foregut microbiome. Nucleotide composition-based sequence binning facilitated the assemblage of more than two megabase pairs of genomic sequence for one of the novel Lachnospiraceae clades (WG-2). These analyses show that WG-2 possesses numerous glycoside hydrolases targeting noncellulosic polysaccharides. These collective data demonstrate that Australian macropods not only harbor unique bacterial lineages underpinning plant biomass conversion, but their repertoire of glycoside hydrolases is distinct from those of the microbiomes of higher termites and the bovine rumen.

Herbivores retain within their gastrointestinal tract a microbiome that specializes in the rapid hydrolysis and fermentation of lignocellulosic plant biomass. With the emergence of high-throughput DNA sequencing technologies and related 'omics' approaches, along with demands to better utilize lignocellulose materials as a feedstock for second-generation biofuels, these gut microbiomes are thought to be a potential source of novel biotechnologies relevant to meeting these needs. This review provides an insight into the new findings that have arisen from the (meta)genomic analysis of specialist cellulolytic bacteria and gut microbiomes of herbivorous insects, ruminants, native Australian marsupials, and other obligate herbivores. In addition to there being more of the same in terms of cellulases and cellulosomes, there also appears to be something 'new' in terms of the compositional and functional attributes of the plant cell wall deconstruction systems employed by these bacteria. However, future dissection and capture of useful biotechnologies via metagenomics will need more than the production of data using next generation sequencing technologies.

High molecular weight (HMW) DNA prepared from a toxic freshwater cyanobacterial bloom sample was used to construct a PCR-generated 75-clone, 16S rRNA gene library and a 2850-clone bacterial artificial chromosome (BAC) library. Phylogenetic analysis of the 16S rRNA gene library demonstrated that members of eight phyla of domain Bacteria, which included Cyanobacteria, Actinobacteria, Verrucomicrobium, Bacteriodetes, Planctomycetes, Chloroflexi, Candidate Division OP10 and Alpha-, Beta- and Gammaproteobacteria, were present in the bloom community. Diversity estimates determined from 16S rRNA gene analysis and direct cell counts and morphological identification of phytoplanktons suggested that the bloom community was dominated by members of the genera Aphanizomenon and Cylindrospermopsis, phylum Cyanobacteria. BAC-end sequencing of 37 randomly selected clones and subsequent sequence analysis provided a snapshot of the total bloom community putative metabolic activities. The sequencing of the entire inserts of seven clones (clones designated 578, 67, 142, 543, 905, 1664 and 2089) selected from BAC-end sequence studies resulted in the generation of a total of 144-kb sequence data and in the identification of 130 genes for putative proteins representing at least four phyla, Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria. This is the first report on a snapshot analysis of a limited metagenome of a toxic cyanobacterial freshwater bloom.

Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning., OCOU was supported by a strategic scholarship program to food science research, from Norwegian University of Life Sciences (NMBU) (project 1205051025). CB and part of this work was supported by a contract financed by the Spanish Ministerio de Economia y Competitividad (AGL2013-47420-R). PBP is supported by a grant from the European Research Council (336355-MicroDE). Travels and stays in Spain and Norway for this study were supported by EEA Coordinated Mobility of Researchers NILS Science and Sustainability Project 017-CM-01-2013. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown. Here we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm. By combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.

We demonstrate that two characteristic Sus-like proteins encoded within a polysaccharide utilization locus (PUL) bind strongly to cellulosic substrates and interact with plant primary cell walls. This shows associations between uncultured Bacteroidetes -affiliated lineages and cellulose in the rumen and thus presents new PUL-derived targets to pursue regarding plant biomass degradation.

Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

Microbial community profiles in two parallel CSTR biogas reactors fed with whey permeate and cow manure were investigated. The operating conditions for these two reactors were identical, yet only one of them (R1) showed stable performance, whereas the other (R2) showed a decrease in methane production accompanied by accumulation of propionic acid and, later, acetic acid. This gave a unique opportunity to study the dynamics of the microbial communities in two biogas reactors apparently operating close to the edge of stability. The microbial community was dominated by Bacteroidetes and Firmicutes, and the methanogens Methanobacteriales and Methanomicrobiales in both reactors, but with larger fluctuations in R2. Correlation analyses showed that the depletion of propionic acid in R1 and the late increase of acetic acid in R2 was related to several bacterial groups. The biogas production in R1 shows that stable co-digestion of manure and whey can be achieved with reasonable yields.

Reaching a comprehensive understanding of how nature solves the problem of degrading recalcitrant biomass may eventually allow development of more efficient biorefining processes. Here we interpret genomic and proteomic information generated from a cellulolytic microbial consortium (termed F1RT) enriched from soil. Analyses of reconstructed bacterial draft genomes from all seven uncultured phylotypes in F1RT indicate that its constituent microbes cooperate in both cellulose-degrading and other important metabolic processes. Support for cellulolytic inter-species cooperation came from the discovery of F1RT microbes that encode and express complimentary enzymatic inventories that include both extracellular cellulosomes and secreted free-enzyme systems. Metabolic reconstruction of the seven F1RT phylotypes predicted a wider genomic rationale as to how this particular community functions as well as possible reasons as to why biomass conversion in nature relies on a structured and cooperative microbial community.

Background Efficient industrial processes for converting plant lignocellulosic materials into biofuels are a key to global efforts to come up with alternative energy sources to fossil fuels. Novel cellulolytic enzymes have been discovered in microbial genomes and metagenomes of microbial communities. However, the identification of relevant genes without known homologs, and the elucidation of the lignocellulolytic pathways and protein complexes for different microorganisms remain challenging. Results We describe a new computational method for the targeted discovery of functional modules of plant biomass-degrading protein families, based on their co-occurrence patterns across genomes and metagenome datasets, and the strength of association of these modules with the genomes of known degraders. From approximately 6.4 million family annotations for 2,884 microbial genomes, and 332 taxonomic bins from 18 metagenomes, we identified 5 functional modules that are distinctive for plant biomass degraders, which we term “plant biomass degradation modules” (PDMs). These modules incorporate protein families involved in the degradation of cellulose, hemicelluloses, and pectins, structural components of the cellulosome, and additional families with potential functions in plant biomass degradation. The PDMs were linked to 81 gene clusters in genomes of known lignocellulose degraders, including previously described clusters of lignocellulolytic genes. On average, 70% of the families of each PDM were found to map to gene clusters in known degraders, which served as an additional confirmation of their functional relationships. The presence of a PDM in a genome or taxonomic metagenome bin furthermore allowed us to accurately predict the ability of any particular organism to degrade plant biomass. For 15 draft genomes of a cow rumen metagenome, we used cross-referencing to confirmed cellulolytic enzymes to validate that the PDMs identified plant biomass degraders within a complex microbial community. Conclusions Functional modules of protein families that are involved in different aspects of plant cell wall degradation can be inferred from co-occurrence patterns across (meta-)genomes with a probabilistic topic model. PDMs represent a new resource of protein families and candidate genes implicated in microbial plant biomass degradation. They can also be used to predict the plant biomass degradation ability for a genome or taxonomic bin. The method is also suitable for characterizing other microbial phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s13068-014-0124-8) contains supplementary material, which is available to authorized users.

Several unique Sus-like polysaccharide utilization loci (PULs) were identified from bacteria resident in bovine rumen microbiomes through functional screening of a fosmid library. The loci were phylogenetically assigned to the genus Prevotella within the phylum Bacteroidetes. These findings were augmented by a bioinformatic re-evaluation of ruminal Prevotella genomes, revealing additional loci not previously reported in the literature. Analysis of Bacteroidales-affiliated genomes reconstructed from a bovine rumen metagenome in a previous study further expanded the diversity of Sus-like PULs resident in this microbiome. Our findings suggest that Sus-like systems represent an important mechanism for degradation of a range of plant-derived glycans in ruminants.

The microorganisms living in our gut have been a black box to us for a long time. However, with the recent advances in high throughput DNA sequencing technologies, it is now possible to assess virtually all microorganisms in our gut including non-culturable ones. With the use of powerful bioinformatics tools to deal with multivariate analyses of huge amounts of data from metagenomics, metatranscriptomics, metabolomics, we now start to gain some important insights into these tiny gut inhabitants. Our knowledge is increasing about who they are, to some extent, what they do and how they affect our health. Gut microbiota have a broad spectrum of possible effects on health, from preventing serious diseases, improving immune system and gut health to stimulating the brain centers responsible for appetite and food intake control. Further, we may be on the verge of being capable of manipulating the gut microbiota by diet control to possibly improve our health. Diets consisting of different components that are fermentable by microbiota are substrates for different kinds of microbes in the gut. Thus, diet control can be used to favor the growth of some selected gut inhabitants. Nowadays, the gut microbiota is taken into account as a separate organ in human body and their activities and metabolites in gut have many physiological and neurological effects. In this mini-review, we discuss the diversity of gut microbiota, the technologies used to assess them, factors that affect microbial composition and metabolites that affect human physiology, and their potential applications in satiety control via the gut-brain axis.

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the "repeated bead beating and column (RBB+C) purification" method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions. Refereed/Peer-reviewed

High molecular weight (HMW) DNA prepared from a toxic freshwater cyanobacterial bloom sample was used to construct a PCR-generated 75-clone, 16S rRNA gene library and a 2850-clone bacterial artificial chromosome (BAC) library. Phylogenetic analysis of the 16S rRNA gene library demonstrated that members of eight phyla of domain Bacteria, which included Cyanobacteria, Actinobacteria, Verrucomicrobium, Bacteriodetes, Planctomycetes, Chloroflexi, Candidate Division OP10 and Alpha-, Beta- and Gammaproteobacteria, were present in the bloom community. Diversity estimates determined from 16S rRNA gene analysis and direct cell counts and morphological identification of phytoplanktons suggested that the bloom community was dominated by members of the genera Aphanizomenon and Cylindrospermopsis, phylum Cyanobacteria. BAC-end sequencing of 37 randomly selected clones and subsequent sequence analysis provided a snapshot of the total bloom community putative metabolic activities. The sequencing of the entire inserts of seven clones (clones designated 578, 67, 142, 543, 905, 1664 and 2089) selected from BAC-end sequence studies resulted in the generation of a total of 144-kb sequence data and in the identification of 130 genes for putative proteins representing at least four phyla, Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria. This is the first report on a snapshot analysis of a limited metagenome of a toxic cyanobacterial freshwater bloom.

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.

Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

Seaweed is a highly attractive marine crop for the production of biofuels, due to its rapid growth rate as well as high polysaccharide and low lignin content. One appealing exploitation route is the production of biogas by anaerobic digestion. Interestingly, despite the compositional differences between seaweed and lignocellulosic biomass, available data indicate that conditions and inocula traditionally used for the latter may work well for seaweed. To gain more insight into the underlying microbial processes, we have generated 16S rRNA gene amplicon pyrosequencing data to comparatively describe microbial communities in biogas digesters containing either the seaweed Saccharina latissima or wheat straw. The seaweed digesters gave better biogas yield and a higher relative abundance of core group Methanosaeta-affiliated Archaea. Conversely, variation in biomass had only minor abundance effects towards dominant bacterial lineages and influenced only low-abundant bacterial OTUs. Affiliations between dominant archaeal and bacterial phylotypes described here and previously identified anaerobic digestion core groups indicate that trends are beginning to emerge within these newly explored microbial ecosystems, the understanding of which is currently impeded by limited published datasets.