This study describes and validates a new method for metagenomic biomarker discovery by way of class comparison, tests of biological consistency and effect size estimation. This addresses the challenge of finding organisms, genes, or pathways that consistently explain the differences between two or more microbial communities, which is a central problem to the study of metagenomics. We extensively validate our method on several microbiomes and a convenient online interface for the method is provided at http://huttenhower.sph.harvard.edu/lefse/.

Metagenomic biomarker discovery and explanation

Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane (CH4). Research on methanogens in the rumen has attracted great interest in the last decade because CH4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.

/pdf/rumen-methanogens-and-mitigation-of-methane-emission-by-anti-4r6c11m3qp.pdf

Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

A better understanding of global ruminal microbiota and metabolites under extensive feeding conditions is a prerequisite for optimizing rumen function and improving ruminant feed efficiency. Furthermore, the gap between the information on the ruminal microbiota and metabolites needs to be bridged. The aim of this study was to investigate the effects of a wide range of forage to concentrate ratios (F:C) on changes and interactions of ruminal microbiota and metabolites. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and Illumina MiSeq sequencing and gas chromatography time-of-ﬂight/mass spectrometry were used to investigate the profile changes of the ruminal microbes and metabolites, and the interaction between them. The predominant bacterial phyla in the rumen were Bacteroidetes (57.2 ± 2.6%) and Firmicutes (26.8 ± 1.6%), and the predominant anaerobic fungi were Neocallimastigomycota (64.3 ± 3.8%) and Ascomycota (22.6 ± 2.4%). In total, 44, 9, 25, and 2 genera, respectively, were identified as the core rumen bacteria, ciliate protozoa, anaerobic fungi, and archaea communities across all samples. An increased concentrate level linearly decreased the relative abundance of cellulolytic bacteria and ciliates, namely Fibrobacter, Succinimonas, Polyplastron, and Ostracodinium (q < 0.05), and linearly increased the relative abundance of Entodinium (q = 0.04), which is a non-fibrous carbohydrate degrader. Dietary F:C had no effect on the communities of anaerobic fungi and archaea. Rumen metabolomics analysis revealed that ruminal amino acids, lipids, organic acids, and carbohydrates were altered significantly by altering the dietary F:C. With increasing dietary concentrate levels, the proportions of propionate and butyrate linearly increased in the rumen (P ≤ 0.01). Correlation analysis revealed that there was some utilization relationship or productive association between candidate metabolites and affected microbe groups. This study provides a better understanding of ruminal microbiota and metabolites under a wide range of dietary F:C, which could further reveal integrative information of rumen function and lead to an improvement in ruminant production.

/pdf/effect-of-dietary-forage-to-concentrate-ratios-on-dynamic-2tvwn0z7dg.pdf

Effect of Dietary Forage to Concentrate Ratios on Dynamic Profile Changes and Interactions of Ruminal Microbiota and Metabolites in Holstein Heifers.

The biological significance of Archaea in the human gut microbiota is largely unclear. We recently reported genomic and biochemical analyses of the Methanomassiliicoccales, a novel order of methanogenic Archaea dwelling in soil and the animal digestive tract. We now show that these Methanomassiliicoccales are present in published microbiome data sets from eight countries. They are represented by five Operational Taxonomic Units present in at least four cohorts and phylogenetically distributed into two clades. Genes for utilizing trimethylamine (TMA), a bacterial precursor to an atherosclerogenic human metabolite, were present in four of the six novel Methanomassiliicoccales genomes assembled from ELDERMET metagenomes. In addition to increased microbiota TMA production capacity in long-term residential care subjects, abundance of TMA-utilizing Methanomassiliicoccales correlated positively with bacterial gene count for TMA production and negatively with fecal TMA concentrations. The two large Methanomassiliicoccales clades have opposite correlations with host health status in the ELDERMET cohort and putative distinct genomic signatures for gut adaptation.

/pdf/genomics-and-metagenomics-of-trimethylamine-utilizing-53ykgb28n0.pdf

Genomics and metagenomics of trimethylamine-utilizing Archaea in the human gut microbiome.

Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane.

Anaerobic fungi (phylum Neocallimastigomycota), an early branching family of fungi, are commonly encountered in the digestive tract of mammalian herbivores. To date, isolates from ten described genera have been reported, and several novel taxonomic groupings are detected using culture-independent molecular methods. Anaerobic fungi are recognized as playing key roles in the decomposition of lignocellulose (up to 50% of the ingested and untreated lignocellulose), with their physical penetration and extracellular enzymatical secretion of an unbiased diverse repertoire of cell-wall-degrading enzymes. The secreted cell-wall-degrading enzymes of anaerobic fungi include both free enzymes and extracellular multi-enzyme complexes called cellulosomes, both of which have potential as fiber degraders in industries. In addition, anaerobic fungi can provide large amounts of substrates such as hydrogen, formate, and acetate for their co-cultured methanogens. Consequently, large amounts of methane can be produced. And thus, it is promising to use the co-culture of anaerobic fungi and methanogens in the biogas process to intensify the biogas yield owing to the efficient and robust degradation of recalcitrant biomass by anaerobic fungi and improved methane production from co-cultures of anaerobic fungi and methanogens.

The biotechnological potential of anaerobic fungi on fiber degradation and methane production

Anaerobic fungi are potent lignocellulose degraders, but have not yet been exploited in this capacity, largely owing to their poor metabolic characterization. In the current study, a time course of fermentation was conducted to study the effect of the co-cultured methanogens on xylose metabolism by anaerobic fungi. The fermentation end-products from anaerobic fungal monoculture were H2 (6.7 ml), CO2 (65.7 ml), formate (17.90 mM), acetate (9.00 mM), lactate (11.89 mM), ethanol, and malate after 96 h fermentation. Compared to the monoculture, the end-products of co-culture shifted to more CO2 (71.8 ml) and acetate (15.20 mM), methane (14.9 ml), less lactate (5.28 mM), and hardly detectable formate and H2 at the end of fermentation. After 48 h, accumulated formate was remarkably consumed by co-cultured methanogens, accompanied by significantly increased acetate, CO2 and pH, and decreased lactate and malate. Xylose utilization, in both cultures, was similar during fermentation. However, the relative flux of carbon in hydrogenosomes in the co-culture was higher than that in the monoculture. In conclusion, the co-culture with methanogens enhanced “energy yields” of anaerobic fungi by removing the accumulated formate, decreased the metabolism in cytosol, for example, the lactate pathway, and increased the metabolism in hydrogenosomes, for example, the acetate pathway.

Indigenously associated methanogens intensified the metabolism in hydrogenosomes of anaerobic fungi with xylose as substrate.

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and α-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and α-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.

/pdf/production-of-citrate-by-anaerobic-fungi-in-the-presence-of-2idhv2x7nc.pdf

Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by (1)H NMR Spectrometry.

Although the scheme of metabolic pathways involved in the production of the major end products has been described, the dynamic profile of metabolites of anaerobic fungi co-cultured with methanogens is limited, especially for the intermediate metabolites. In the present study, the fermentation of the co-culture of Piromyces sp. F1 and Methanobrevibacter thaueri on glucose was investigated. The presence of methanogens shortened the growth lag time of anaerobic fungi and enhanced the total gas production. The occurrence of the maximum cell dry weight and the disappearance of most of the substrate were observed at 24 h for the co-culture and 48 h for the fungal mono-culture. In the co-culture, hydrogen was detected at a very low level during fermentation, and formate transitorily accumulated at 24 h and disappeared at 48 h, resulting in an increase of pH. Acetate was higher during the fermentation in the co-culture (P < 0.05), while lactate and ethanol were higher only in the initial stage of fermentation (P < 0.05). After 48 h, lactate in the mono-culture became much higher than that in the co-culture (P < 0.05), and ethanol tended to remain the same in both cultures. Moreover, malate tended to be exhausted in the co-culture, while it accumulated in the mono-culture. Citrate was also detected in both co-culture and mono-culture. Collectively, these results suggest that methanogen enhanced the malate pathway and weakened the lactate pathway of anaerobic fungus.

Effect of the Associated Methanogen Methanobrevibacter thaueri on the Dynamic Profile of End and Intermediate Metabolites of Anaerobic Fungus Piromyces sp. F1

Wei Jin

Papers

Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane.

The biotechnological potential of anaerobic fungi on fiber degradation and methane production

Indigenously associated methanogens intensified the metabolism in hydrogenosomes of anaerobic fungi with xylose as substrate.

Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by (1)H NMR Spectrometry.

Effect of the Associated Methanogen Methanobrevibacter thaueri on the Dynamic Profile of End and Intermediate Metabolites of Anaerobic Fungus Piromyces sp. F1