The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial ...degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F succinogenes rumen strain S85 has recently suggested that within this group of organisms a "third" way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.
Accurate quantification of biodiversity is fundamental to understanding ecosystem function and for environmental assessment. Molecular methods using environmental DNA (eDNA) offer a non-invasive, ...rapid, and cost-effective alternative to traditional biodiversity assessments, which require high levels of expertise. While eDNA analyses are increasingly being utilized, there remains considerable uncertainty regarding the dynamics of multispecies eDNA, especially in variable systems such as rivers. Here, we utilize four sets of upland stream mesocosms, across an acid-base gradient, to assess the temporal and environmental degradation of multispecies eDNA. Sampling included water column and biofilm sampling over time with eDNA quantified using qPCR. Our findings show that the persistence of lotic multispecies eDNA, sampled from water and biofilm, decays to non-detectable levels within 2 days and that acidic environments accelerate the degradation process. Collectively, the results provide the basis for a predictive framework for the relationship between lotic eDNA degradation dynamics in spatio-temporally dynamic river ecosystems.
Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon ...microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.
The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is ...critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) "baits" were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of
,
,
, and
in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial "cellulosome" systems of members of the
, we report the first detection of the
cellulase system and the
polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance.
The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, we identified
,
, and
as key phyla in the landfill cellulolytic community, detecting 8,371 carbohydrate active enzymes (CAZymes) that represent at least three of the recognized strategies for cellulose decomposition. These data highlight substantial hydrolytic enzyme diversity in landfill sites as a source of new enzymes for biomass conversion.
Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, ...Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch's postulates to address the role of microbial communities in disease.
The Fibrobacteres phylum contains two described species, Fibrobacter succinogenes and Fibrobacter intestinalis, both of which are prolific degraders of cellulosic plant biomass in the herbivore gut. ...However, recent 16S rRNA gene sequencing studies have identified novel Fibrobacteres in landfill sites, freshwater lakes and the termite hindgut, suggesting that members of the Fibrobacteres occupy a broader ecological range than previously appreciated. In this study, the ecology and diversity of Fibrobacteres was evaluated in 64 samples from contrasting environments where cellulose degradation occurred. Fibrobacters were detected in 23 of the 64 samples using Fibrobacter genus-specific 16S rRNA gene PCR, which provided their first targeted detection in marine and estuarine sediments, cryoconite from Arctic glaciers, as well as a broader range of environmental samples. To determine the phylogenetic diversity of the Fibrobacteres phylum, Fibrobacter-specific 16S rRNA gene clone libraries derived from 17 samples were sequenced (384 clones) and compared with all available Fibrobacteres sequences in the Ribosomal Database Project repository. Phylogenetic analysis revealed 63 lineages of Fibrobacteres (95% OTUs), with many representing as yet unclassified species. Of these, 24 OTUs were exclusively comprised of fibrobacters derived from environmental (non-gut) samples, 17 were exclusive to the mammalian gut, 15 to the termite hindgut, and 7 comprised both environmental and mammalian strains, thus establishing Fibrobacter spp. as indigenous members of microbial communities beyond the gut ecosystem. The data highlighted significant taxonomic and ecological diversity within the Fibrobacteres, a phylum circumscribed by potent cellulolytic activity, suggesting considerable functional importance in the conversion of lignocellulosic biomass in the biosphere.
Under optimized conditions, the lactic acid bacterium Lactobacillus fermentum Lf2 secretes up to 2 gL-1 of a mixture of polysaccharides into the fermentation medium when grown on sucrose. Earlier ...studies had shown that the mixture is biologically active and work was undertaken to characterise the polysaccharides. Preparative size exclusion chromatography was used to separate a high molecular mass β-glucan (weight average mass of 1.23 × 106 gmol−1) from two medium molecular mass polysaccharides (weight average mass of 8.8 × 104 gmol−1). Under optimized growth conditions, the medium molecular mass polysaccharides accounted for more than 75% of the mixture by weight. Monomer, linkage analysis and NMR spectroscopy of the medium molecular mass polysaccharides, and material isolated after their Smith degradation, was used to identify the structure of the component polysaccharides. The mixture contains two novel polysaccharides. The first has a main chain of β-1,6-linked galactofuranoses which is non-stoichiometrically 2-O-glucosylated. The degree of substitution at the 2-position, with α-D-Glcp, depends on the fermentation conditions; under optimized conditions greater than 80% 2-O-α-D-glucosylation was observed.
The second polysaccharide is a heteroglycan with four monosaccharides in the repeat unit: residual signals in the NMR suggest that the sample also contains trace amounts (<3%) of cell wall polysaccharides.
Display omitted
•Isolation of two medium molecular mass exopolysaccharides from L fermentum Lf2.•Characterization of both medium molecular mass exopolysaccharides (MMMP 1 & 2).•MMMP1 has a main chain of 1,6-β-D-Galfs with non-stoichiometric 2-O-glucosylation.•MMMP2 has a repeat unit .→3)-β-D-Glcp-(1 → 3)-β-D-Galf-(1 → 6)-2-α-D-Glcpβ-D-Galf-(1→
Here, we report the draft genome of Clostridium sp. strain W14A, isolated from the anaerobic, cellulolytic biofilm of a cotton string sample incubated in a landfill leachate microcosm. The draft ...genome comprises 131 contigs, 3,823,510 bp, 51.5% G+C content, and 4,119 predicted coding domain sequences.
The Fibrobacteres phylum contains only two characterised species, Fibrobacter succinogenes and F. intestinalis, both of which are important degraders of cellulose in the mammalian gut. Fibrobacters ...were thought to be restricted to the gut environment, but this was recently disproven via the detection of novel Fibrobacter spp. in landfill sites and freshwater lakes, with the implication that the distribution and diversity of members of the genus Fibrobacter is poorly understood. Here, the environmental distribution of members of the genus Fibrobacter was addressed; a total of 64 samples from contrasting environments were screened for the presence of Fibrobacter spp. via genus-specific 16S rRNA gene PCR primers. Fibrobacters were detected in 23 samples, with the first specific detection of fibrobacters in marine and estuarine sediments, and Arctic cryoconite. Phylogenetic analysis of 16S rRNA gene sequences revealed 63 Fibrobacter OTUs at 95% sequence similarity, representing a wealth of unclassified species contained within this genus. To address the lack of cultivated Fibrobacter isolates, their isolation from landfill leachate microcosms containing either Avicel or dewaxed cotton string as the sole carbon source for growth was attempted, resulting in the first isolation of F. succinogenes from a landfill site, and the first isolation of fibrobacters from outside the mammalian gut. This is the first phenotypic evidence that fibrobacters are active members of the landfill cellulolytic community. The importance of fibrobacters in landfill cellulose decomposition was further characterised via 454 pyrosequencing of 16S rRNA gene amplicons and shotgun metagenomic sequencing of heavily-degraded cotton samples from landfill leachate microcosm, where fibrobacters have previously been shown to predominate. Fibrobacter 16S rRNA gene amplicons were enriched on the heavily degraded cotton sample (14.2% of sequences, vs. 0.02% for a poorly degraded cotton sample), and metagenome sequencing of the heavily degraded cotton revealed that proteins most closely related to F. succinogenes strain S85 dominated the sequence output. Attempts to isolate Fibrobacter spp. from the same heavily degraded cotton sample revealed that Fibrobacter spp. were present in co-culture with Clostridium sporogenes, but attempts to purify these strains were unsuccessful. These data represent a significant contribution to our understanding of the ecology of members of the genus Fibrobacter, and their important role in the hydrolysis of cellulolytic biomass.
PDF
