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Disposal of electrons generated during the fermentation of ingested feed is a fundamental feature of anaerobic microbial gut ecosystems. Here, we focus on the well-studied rumen environment to highlight how electrons are transferred through anaerobic fermentation pathways and how manipulating this electron flow is important to reducing methane emissions from ruminants. Priorities for research that can accelerate understanding in this area are highlighted.

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 hours in nylon bags within the rumen of cannulated dairy cows. Across a gene catalogue covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of polysaccharides such as hemicellulose, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8 and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks ofin siturecalcitrant fiber deconstruction, and importantly, suggests that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown etiology; however, apoptosis of lung alveolar epithelial cells plays a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis remain elusive. Here, we report that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. Our results suggest that bacteria shedding corisin are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of staphylococci in pulmonary fibrosis., Idiopathic pulmonary fibrosis is associated with increased abundance of Staphylococcus and Streptococcus in the lungs. Here, the authors identify a Staphylococcus nepalensis-derived peptide, named corisin, to induce apoptosis of lung epithelial cells and exacerbation of pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Acute exacerbation of idiopathic pulmonary fibrosis is associated with high mortality. Excessive apoptosis of lung epithelial cells occurs in pulmonary fibrosis acute exacerbation. We recently identified corisin, a proapoptotic peptide that triggers acute exacerbation of pulmonary fibrosis. Here, we provide insights into the mechanism underlying the processing and release of corisin. Furthermore, we demonstrate that an anticorisin monoclonal antibody ameliorates lung fibrosis by significantly inhibiting acute exacerbation in the human transforming growth factorβ1 model and acute lung injury in the bleomycin model. By investigating the impact of the anticorisin monoclonal antibody in a general model of acute lung injury, we further unravel the potential of corisin to impact such diseases. These results underscore the role of corisin in the pathogenesis of acute exacerbation of pulmonary fibrosis and acute lung injury and provide a novel approach to treating this incurable disease.

Fully understanding the dynamic distribution of the gut microbiota in pigs is essential, as gut microorganisms play a fundamental role in physiological processes, immunity, and the metabolism of nutrients by the host. Here, we first summarize the characteristics and the dynamic shifts in the gut microbial community of pigs at different ages based on the results of 63 peer-review publications. Then a meta-analysis based on the sequences from 16 studies with accession numbers in the GenBank database is conducted to verify the characteristics of the gut microbiota in healthy pigs. A dynamic shift is confirmed in the gut microbiota of pigs at different ages and growth phases. In general, Bacteroides, Escherichia, Clostridium, Lactobacillus, Fusobacterium, and Prevotella are dominant in piglets before weaning, then Prevotella and Aneriacter shift to be the predominant genera with Fusobacterium, Lactobacillus, and Miscellaneous as comparative minors in postweaned pigs. A number of 19 bacterial genera, including Bacteroides, Prevotella, and Lactobacillus can be found in more than 90% of pigs and three enterotypes can be identified in all pigs at different ages, suggesting there is a “core” microbiota in the gut of healthy pigs, which can be a potential target for nutrition or health regulation. The “core” members benefit the growth and gut health of the host. These findings help to define an “optimal” gut microbial profile for assessing, or improving, the performance and health status of pigs at different growth stages. IMPORTANCE The ban on feed antibiotics by more and more countries, and the expected ban on ZnO in feed supplementation from 2022 in the EU, urge researchers and pig producers to search for new alternatives. One possible alternative is to use the so-called “next-generation probiotics (NGPs)” derived from gastrointestinal tract. In this paper, we reveal that a total of 19 “core” bacterial genera including Bacteroides, Prevotella, and Lactobacillus etc., can be found in more than 90% of healthy pigs across different ages. These identified genera may probably be the potential candidates of NGPs or the potential target of microflora regulation. Adding substrates preferred by these target microbes will help to increase the abundance of specific symbiotic species and benefit the gut health of pigs. Further research targeting these “core” microbes and the dynamic distribution of microbiota, as well as the related function is of great importance in swine production.

Molecular hydrogen (H2) and formate (HCOO−) are metabolic end products of many primary fermenters in the mammalian gut. Both play a vital role in fermentation where they are electron sinks for individual microbes in an anaerobic environment that lacks external electron acceptors. If H2 and/or formate accumulate within the gut ecosystem, the ability of primary fermenters to regenerate electron carriers may be inhibited and microbial metabolism and growth disrupted. Consequently, H2- and/or formate-consuming microbes such as methanogens and homoacetogens play a key role in maintaining the metabolic efficiency of primary fermenters. There is increasing interest in identifying approaches to manipulate mammalian gut environments for the benefit of the host and the environment. As H2 and formate are important mediators of interspecies interactions, an understanding of their production and utilisation could be a significant entry point for the development of successful interventions. Ruminant methane mitigation approaches are discussed as a model to help understand the fate of H2 and formate in gut systems.

Background Digestive processes in the rumen lead to the release of methyl-compounds, mainly methanol and methylamines, which are used by methyltrophic methanogens to form methane, an important agricultural greenhouse gas. Methylamines are produced from plant phosphatidylcholine degradation, by choline trimethylamine lyase, while methanol comes from demethoxylation of dietary pectins via pectin methylesterase activity. We have screened rumen metagenomic and metatranscriptomic datasets, metagenome assembled genomes, and the Hungate1000 genomes to identify organisms capable of producing methyl-compounds. We also describe the enrichment of pectin-degrading and methane-forming microbes from sheep rumen contents and the analysis of their genomes via metagenomic assembly. Results Screens of metagenomic data using the protein domains of choline trimethylamine lyase (CutC), and activator protein (CutD) found good matches only to Olsenella umbonata and to Caecibacter, while the Hungate1000 genomes and metagenome assembled genomes from the cattle rumen found bacteria within the phyla Actinobacteria, Firmicutes and Proteobacteria. The cutC and cutD genes clustered with genes that encode structural components of bacterial microcompartment proteins. Prevotella was the dominant genus encoding pectin methyl esterases, with smaller numbers of sequences identified from other fibre-degrading rumen bacteria. Some large pectin methyl esterases (> 2100 aa) were found to be encoded in Butyrivibrio genomes. The pectin-utilising, methane-producing consortium was composed of (i) a putative pectin-degrading bacterium (phylum Tenericutes, class Mollicutes), (ii) a galacturonate-using Sphaerochaeta sp. predicted to produce acetate, lactate, and ethanol, and (iii) a methylotrophic methanogen, Methanosphaera sp., with the ability to form methane via a primary ethanol-dependent, hydrogen-independent, methanogenesis pathway. Conclusions The main bacteria that produce methyl-compounds have been identified in ruminants. Their enzymatic activities can now be targeted with the aim of finding ways to reduce the supply of methyl-compound substrates to methanogens, and thereby limit methylotrophic methanogenesis in the rumen.

Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H2 metabolism. Two-thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyse H2 production or consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g., Ruminococcus) accounted for half of all hydrogenase transcripts. Various H2 uptake pathways were also expressed, including methanogenesis (Methanobrevibacter), fumarate and nitrite reduction (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis-related transcripts predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic fumarate reducer Wolinella succinogenes in co-culture compared with pure culture. We conclude that H2 metabolism is a more complex and widespread trait among rumen microorganisms than previously recognised. There is evidence that alternative hydrogenotrophs, including acetogenic and respiratory bacteria, can prosper in the rumen and effectively compete with methanogens for H2. These findings may help to inform ongoing strategies to mitigate methane emissions by increasing flux through alternative H2 uptake pathways, including through animal selection, dietary supplementation and methanogenesis inhibitors.

Background Sucrose is an attractive industrial carbon source due to its abundance and the fact that it can be cheaply generated from sources such as sugarcane. However, only a few characterized Escherichia coli strains are able to metabolize sucrose, and those that can are typically slow growing or pathogenic strains. Methods To generate a platform strain capable of efficiently utilizing sucrose with a high growth rate, adaptive laboratory evolution (ALE) was utilized to evolve engineered E. coli K-12 MG1655 strains containing the sucrose utilizing csc genes (cscB, cscK, cscA) alongside the native sucrose consuming E. coli W. Results Evolved K-12 clones displayed an increase in growth and sucrose uptake rates of 1.72- and 1.40-fold on sugarcane juice as compared to the original engineered strains, respectively, while E. coli W clones showed a 1.4-fold increase in sucrose uptake rate without a significant increase in growth rate. Whole genome sequencing of evolved clones and populations revealed that two genetic regions were frequently mutated in the K-12 strains; the global transcription regulatory genes rpoB and rpoC, and the metabolic region related to a pyrimidine biosynthetic deficiency in K-12 attributed to pyrE expression. These two mutated regions have been characterized to confer a similar benefit when glucose is the main carbon source, and reverse engineering revealed the same causal advantages on M9 sucrose. Additionally, the most prevalent mutation found in the evolved E. coli W lineages was the inactivation of the cscR gene, the transcriptional repression of sucrose uptake genes. Conclusion The generated K-12 and W platform strains, and the specific sets of mutations that enable their phenotypes, are available as valuable tools for sucrose-based industrial bioproduction in the facile E. coli chassis. Electronic supplementary material The online version of this article (10.1186/s12934-019-1165-2) contains supplementary material, which is available to authorized users.

Bacteroides spp. of the human colonic microbiome degrade complex arabinoxylans from dietary fiber and release ferulic acid. Several studies have demonstrated the beneficial effects of ferulic acid. Here, we hypothesized that ferulic acid or the ferulic acid-rich culture supernatant of Bacteroides intestinalis, cultured in the presence of complex arabinoxylans, enhances the immune response. Ferulic acid and the culture supernatant of bacteria cultured in the presence of insoluble arabinoxylans significantly decreased the expression of tumor necrosis factor-α and increased the expression of interleukin-10 and transforming growth factor β1 from activated dendritic cells compared to controls. The number of granulocytes in mesenteric lymph nodes, the number of spleen monocytes/granulocytes, and interleukin-2 and interleukin-12 plasma levels were significantly increased in mice treated with ferulic acid or the culture supernatant of bacteria cultured with insoluble arabinoxylans. Ferulic acid or the culture supernatant of bacteria cultured with insoluble arabinoxylans increased the expression of interleukin-12, interferon-α, and interferon-β in intestinal epithelial cell lines. This study shows that ferulic acid or the ferulic acid-rich culture supernatant of the colonic bacterium Bacteroides intestinalis, cultured with insoluble arabinoxylans, exerts anti-inflammatory activity in dendritic cells under inflammatory conditions and enhances the Th1-type immune response under physiological conditions in mice.

Bacteroides spp. of the human colonic microbiome degrade complex arabinoxylans from dietary fiber and release ferulic acid. Several studies have demonstrated the beneficial effects of ferulic acid. Here, we hypothesized that ferulic acid or the ferulic acid-rich culture supernatant of Bacteroides intestinalis, cultured in the presence of complex arabinoxylans, enhances the immune response. Ferulic acid and the culture supernatant of bacteria cultured in the presence of insoluble arabinoxylans significantly decreased the expression of tumor necrosis factor-α and increased the expression of interleukin-10 and transforming growth factor β1 from activated dendritic cells compared to controls. The number of granulocytes in mesenteric lymph nodes, the number of spleen monocytes/granulocytes, and interleukin-2 and interleukin-12 plasma levels were significantly increased in mice treated with ferulic acid or the culture supernatant of bacteria cultured with insoluble arabinoxylans. Ferulic acid or the culture supernatant of bacteria cultured with insoluble arabinoxylans increased the expression of interleukin-12, interferon-α, and interferon-β in intestinal epithelial cell lines. This study shows that ferulic acid or the ferulic acid-rich culture supernatant of the colonic bacterium Bacteroides intestinalis, cultured with insoluble arabinoxylans, exerts anti-inflammatory activity in dendritic cells under inflammatory conditions and enhances the Th1-type immune response under physiological conditions in mice.

Anaerobiosis is a well-established biotechnology which treats animal manure for methanogenesis. The presence of various antibiotics in poultry waste may inhibit anaerobic digestion (AD). The current study aimed at investigating the effects of various antibiotics on the AD of poultry litter. The batch experiments were performed with an inoculum/substrate ratio of 1 in sealed glass vials incubated under anaerobic conditions at 37 °C for 21 days. The antibiotics, i.e., bacitracin (BAC), erythromycin (ERY), monensin (MON), and tetracycline (TET), were added at the concentration ranges of 0.02 to 0.1 mg mL−1. During the first week, the inhibitory effects of monensin were higher (36–60%) as compared with other antibiotics. For 8–15 days and 16–21 days, the inhibitory effects caused by erythromycin were 28–38%, followed by 21–35% of TET and 20–40% of BAC. MON showed lower inhibition during the final stages of experiment which might possibly be due to its biodegradation.

Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment.

Thermoanaerobacterium bryantii strain mel9T is a thermophilic bacterium isolated from a waste pile of a corn-canning factory. The genome of T. bryantii mel9T was sequenced and a hemicellulase gene cluster was identified. The cluster encodes seven putative enzymes, which are likely an endoxylanase, an α-glucuronidase, two oxidoreductases, two β-xylosidases, and one acetyl xylan esterase. These genes were designated tbxyn10A, tbagu67A, tbheoA, tbheoB, tbxyl52A, tbxyl39A, and tbaxe1A, respectively. Only TbXyn10A released reducing sugars from birchwood xylan, as shown by thin-layer chromatography analysis. The five components of the hemicellulase cluster (TbXyn10A, TbXyl39A, TbXyl52A, TbAgu67A, and TbAxe1A) functioned in synergy to hydrolyze birchwood xylan. Surprisingly, the two putative oxidoreductases increased the enzymatic activities of the gene products from the xylanolytic gene cluster in the presence of NADH and manganese ions. The two oxidoreductases were therefore named Hemicellulase-Enhancing Oxidoreductases (HEOs). All seven enzymes were thermophilic and acted in synergy to degrade xylans at 60 °C. Except for TbXyn10A, the other enzymes encoded by the gene cluster were conserved with high amino acid identities (85–100%) in three other Thermoanaerobacterium species. The conservation of the gene cluster is, therefore, suggestive of an important role of these enzymes in xylan degradation by these bacteria. The mechanism for enhancement of hemicellulose degradation by the HEOs is under investigation. It is anticipated, however, that the discovery of these new actors in hemicellulose deconstruction will have a significant impact on plant cell wall deconstruction in the biofuel industry.

Summary Different factors may modulate the gut microbiota of animals. In any particular environment, diet, genetic factors, and human influences can shape the bacterial communities residing in the gastrointestinal tract. Metagenomic approaches have significantly expanded our knowledge on microbiota dynamics inside hosts, yet cultivation and isolation of bacterial members of these complex ecosystems may still be necessary to fully understand interactions between bacterial communities and their host. A dual approach, involving culture-independent and -dependent techniques, was employed here to decipher the microbiota communities that inhabit the gastro intestinal tract of free-range, broiler and feral chickens. In silico analysis revealed the presence of a core microbiota that is typical of those animals that live in different geographical areas and that have limited contact with humans. Anthropic influences guide the metabolic potential and the presence of antibiotic resistance genes of these different bacterial communities. Culturomics attempts, based on different cultivation conditions, were applied to reconstruct in vitro the microbiota of feral chickens. A unique strain collection representing members of the four major phyla of the poultry microbiota was assembled, including bacterial strains that are not typically retrieved from the chicken gut. This article is protected by copyright. All rights reserved.

Nitrogen metabolism in gut systems remains poorly studied in spite of its importance for microbial growth and its implications for the metabolism of the host. Prevotella spp. are the most predominant bacteria detected in the rumen, but their presence has also been related to health and disease states in the human gut and oral cavity. To explore the metabolic networks for nitrogen assimilation in this bacterium, changes in gene expression profiles in response to variations in the available nitrogen source and to different concentrations of ammonium were analyzed by microarray and reverse transcription quantitative PCR, and linked with function by further proteomic analysis. The observed patterns of transcript abundances for genes involved in ammonium assimilation differed from the classical “enteric paradigm” for nitrogen utilization. Expression of genes encoding high substrate affinity nitrogen assimilation enzymes (GS-GOGAT system) was similar in growth-limiting and non-limiting nitrogen concentrations in P. ruminicola 23, whereas E. coli and Salmonella spp. responses to excess nitrogen involve only low substrate affinity enzymes. This versatile behavior might be a key feature for ecological success in habitats such as the rumen and human colon where nitrogen is rarely limiting for growth, and might be linked to previously reported Prevotella spp. population imbalances relative to other bacterial species in gut systems.

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.

Sugarcane bioethanol has favorable energy and greenhouse gas balance, although the production process generates several residues including vinasse, which deserves attention because of its significant methane (CH

In this study, the effects of a syntrophic methanogen on the growth of Pecoramyces sp. F1 was investigated by characterizing fermentation profiles, as well as functional genomic, transcriptomic, and proteomic analysis. The estimated genome size, GC content, and protein coding regions of strain F1 are 106.83 Mb, 16.07%, and 23.54%, respectively. Comparison of the fungal monoculture with the methanogen co-culture demonstrated that during the fermentation of glucose, the co-culture initially expressed and then down-regulated a large number of genes encoding both enzymes involved in intermediate metabolism and plant cell wall degradation. However, the number of up-regulated proteins doubled at the late-growth stage in the co-culture. In addition, we provide a mechanistic understanding of the metabolism of this fungus in co-culture with a syntrophic methanogen. Further experiments are needed to explore this interaction during degradation of more complex plant cell wall substrates.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis, on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties., Human gut bacteria can degrade arabinoxylans, polysaccharides found in dietary fiber. Here, Pereira et al. identify a bacterial gene cluster encoding esterases for degradation of complex arabinoxylans. The action of these enzymes results in accumulation of ferulic acid, a phenolic compound with antioxidative and immunomodulatory properties.

A reciprocal cross-fostering model with an obese typical Chinese piglet breed and a lean Western breed was used to identify genetic and maternal effects on the acquisition and development gut bacteria from birth until after weaning. Pyrosequencing of 16S rRNA genes results revealed an age- and diet-dependent bacterial succession process in piglets. During the first 3 days after birth, the bacterial community was relatively simple and dominated by Firmicutes with 79% and 65% relative abundance for Meishan and Yorkshire piglets, respectively. During the suckling period until day 14, the piglet breed and the nursing mother lead to increasing differentiation of the fecal bacterial community, with specific bacteria taxa associated with breed, and others with the nursing sow most likely due to its milk composition. Although the effect of nursing mother and the breed were evident through the suckling period, the introduction of solid feed and subsequent weaning were the major events occurring that dominated succession of the gut microbiota in the early life of piglets. This piglet cross-fostering model is a useful tool for studying the effects of diet, host genetics and the environment on the development and acquisition of the gut microbiota and over longer studies the subsequent impact on growth, health and performance of pigs.

Detailed biochemical, structural and growth studies reveal how Bacteroides thetaiotaomicron coordinates a complex enzymatic response to deconstruct pectins — complex dietary components that comprise a tremendous diversity of monosaccharide units and glycosidic linkage combinations.

Sugarcane bioethanol has favorable energy and greenhouse gas balance, although the production process generates several residues including vinasse, which deserves attention because of its significant methane (CH4) emission during storage and transportation stages. Considering that CH4 emissions are dependent on the structure and abundance of microbial communities, we hypothesized that different vinasse transportation systems would harbor different microbial community composition, resulting in distinct CH4 patterns. To test this hypothesis, we used high-throughput 16S rRNA sequencing with real-time PCR to evaluate the composition and abundance of microorganisms in the two main systems of vinasse storage and transportation (i.e. open channels and tanks systems) in Brazil. Our results showed higher microbial diversity and CH4 emissions in channel system, especially in the uncoated section. Significant differences in microbial community structure, diversity, and abundance between the uncoated/coated open channel and tanks indicated a clear selection at taxonomic and functional levels, especially in relation to CH4 production. These responses included higher methanogens diversity in the uncoated section of the channel and are in agreement with the methanogen abundance determined by mcrA and mba genes copy number (1.5 × 107 and 4.3 × 1010) and subsequent positive correlation with CH4 emissions (R2 = 0.8). The most representative methanogen genus across the samples was Methanobrevibacter. The results observed herein shows that the use of the coating in the bottom of channels and tanks prevent the growth and development of a methanogen-related community. We concluded that the improvements in vinasse storage and transportation systems would significantly change the microbial community and reduce CH4 emissions, thereby making bioethanol a greener biofuel.

Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic, and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H2metabolism. Two thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyze H2production or consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g.Ruminococcus) accounted for half of all hydrogenase transcripts. Various H2uptake pathways were also expressed, including methanogenesis (Methanobrevibacter), fumarate reduction and nitrate ammonification (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenterRuminococcus albusand the hydrogenotrophic fumarate reducerWolinella succinogenesin co-culture compared to pure culture. We conclude that H2metabolism is a more complex and widespread trait among rumen microorganisms than previously recognized. There is evidence that alternative hydrogenotrophs, including acetogens and selenomonads, can prosper in the rumen and effectively compete with methanogens for H2in low methane yield ruminants. Strategies to increase flux through alternative H2uptake pathways, including animal selection, dietary supplementation, and methanogenesis inhibitors, may lead to sustained methane mitigation.

Idiopathic pulmonary fibrosis (IPF) is an incurable disease with poor prognosis and unknown etiology. The poor clinical outcome is associated with enhanced microbial burden in bronchoalveolar lavage fluid from IPF patients. However, whether microbes from the respiratory tract fluid cause the disease remains uncertain. Tissue-associated microbes can influence host physiology in health and disease development. The aim of this study was to evaluate the existence of microbes in lung fibrotic tissues. We evaluated the microbial community in lung tissues from IPF and from human transforming growth factor-β1 (TGF-β1) transgenic mice with lung fibrosis by oligotyping. We also evaluated the microbial population in non-tumor-bearing tissues from surgical specimens of lung cancer patients. The phyla Firmicutes and the genus Clostridium tended to be predominant in the lung tissue from IPF and lung cancer patients. Oligotyping analysis revealed a predominance of bacteria belonging to the genera Halomonas, Shewanella, Christensenella, and Clostridium in lung tissue from IPF and lung cancer. Evaluation of the microbial community in the lung tissue from mice revealed abundance of Proteobacteria in both wild-type (WT) littermates and transgenic mice. However, the genus Halomonas tended to be more abundant in TGF-β1 transgenic mice compared to WT mice. In conclusion, this study describes tissue-associated microbes in lung fibrotic tissues from IPF patients and from aging TGF-β1 transgenic mice.

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.

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α- l -arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β- l -arabinopyranosidase (CpAbp27A), and two GH127 β- l -arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para -nitrophenyl ( p NP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α- l -arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l -arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus . IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus . The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β- l -arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98A resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.

For most mammals, including nonhuman primates, diet composition varies temporally in response to differences in food availability. Because diet influences gut microbiota composition, it is likely that the gut microbiota of wild mammals varies in response to seasonal changes in feeding patterns. Such variation may affect host digestive efficiency and, ultimately, host nutrition. In this study, we investigate the temporal variation in diet and gut microbiota composition and function in two groups (N = 13 individuals) of wild Mexican black howler monkeys (Alouatta pigra) over a 10-month period in Palenque National Park, Mexico. Temporal changes in the relative abundances of individual bacterial taxa were strongly correlated with changes in host diet. For example, the relative abundance of Ruminococcaceae was highest during periods when energy intake was lowest, and the relative abundance of Butyricicoccus was highest when young leaves and unripe fruit accounted for 68 % of the diet. Additionally, the howlers exhibited increased microbial production of energy during periods of reduced energy intake from food sources. Because we observed few changes in howler activity and ranging patterns during the course of our study, we propose that shifts in the composition and activity of the gut microbiota provided additional energy and nutrients to compensate for changes in diet. Energy and nutrient production by the gut microbiota appears to provide an effective buffer against seasonal fluctuations in energy and nutrient intake for these primates and is likely to have a similar function in other mammal species.

Hemicelluloses, the polysaccharide component of plant cell walls, represent one of the most abundant biopolymers in nature. The most common hemicellulosic constituents of softwoods, such as conifers and cycads, are mannans consisting of a 1,4-linked β-mannopyranosyl main chain with branch decorations. Efforts toward the utilization of hemicellulose for bioconversion into cellulosic biofuels have resulted in the identification of several families of glycoside hydrolases that can degrade mannan. However, effective biofermentation of manno-oligosaccharides is limited by a lack of appropriate uptake route in ethanologenic organisms. Here, we used transcriptome sequencing to gain insights into mannan degradation by the thermophilic anaerobic bacterium Caldanaerobius polysaccharolyticus. The most highly up-regulated genes during mannan fermentation occur in a cluster containing several genes encoding enzymes for efficient mannan hydrolysis as well as a solute-binding protein (CpMnBP1) that exhibits specificity for short mannose polymers but exhibited the flexibility to accommodate branched polysaccharide decorations. Co-crystal structures of CpMnBP1 in complex with mannobiose (1.4-Å resolution) and mannotriose (2.2-Å resolution) revealed the molecular rationale for chain length and oligosaccharide specificity. Calorimetric analysis of several active site variants confirmed the roles of residues critical to the function of CpMnBP1. This work represents the first biochemical characterization of a mannose-specific solute-binding protein and provides a framework for engineering mannan utilization capabilities for microbial fermentation.

In all mammals, growth, development, pregnancy, and lactation increase nutritional demands. Although primate field studies tend to focus on shifts in activity and diet as mechanisms to compensate for these demands, differences in digestive efficiency also are likely to be important. Because the gut microbiota can impact host digestive efficiency, we examined differences in activity budget, diet, and the gut microbial community among adult male (N = 4), adult female (N = 4), and juvenile (N = 5) wild black howler monkeys (Alouatta pigra) across a ten-month period in Palenque National Park, Mexico to determine how adult females and juveniles compensate for increased nutritional demands. Results indicate that adult females and juveniles consumed more protein and energy than adult males. Adult males, adult females, and juveniles also possessed distinct gut microbial communities, unrelated to diet. Juveniles exhibited a gut microbiota characterized by bacteria from the phylum Firmicutes, such as Roseburia and Ruminococcus, and demonstrated high fecal volatile fatty acid content, suggesting increased microbial contributions to host energy balances. Adult females possessed a higher Firmicutes to Bacteroidetes ratio, also suggesting increased energy production, and their gut microbiota was characterized by Lactococcus, which has been associated with folate biosynthesis. On the basis of these patterns, it appears that the gut microbiota differentially contributes to howler monkey nutrition during reproduction and growth. Determining the nutritional and energetic importance of shifts in activity, diet, and the gut microbiota in other nonhuman primate taxa, as well as humans, will transform our understanding of these life history processes and the role of host-microbe relationships in primate evolution. Am J Phys Anthropol 155:652–664, 2014. © 2014 Wiley Periodicals, Inc.

Identification of microbes that actively utilize root exudates is essential to understand plant-microbe interactions. To identify active root exudate-utilizing microorganisms associated with switchgrass - a potential bioenergy crop - plants were labelled in situ with (13) CO2 , and 16S and 18S rRNA genes in the (13) C-labelled rhizosphere DNA were pyrosequenced. Multi-pulse labelling for 5 days produced detectable (13) C-DNA, which was well separated from unlabelled DNA. Methylibium from the order Burkholderiales were the most heavily labelled bacteria. Pythium, Auricularia and Galerina were the most heavily labelled eukaryotic microbes. We also identified a Glomus intraradices-like species; Glomus members are arbuscular mycorrhizal fungi that are able to colonize the switchgrass root. All of these heavily labelled microorganisms were also among the most abundant species in the rhizosphere. Species belonging to Methylibium and Pythium were the most heavily labelled and the most abundant bacteria and eukaryotes in the rhizosphere of switchgrass. Our results revealed that nearly all of the dominant rhizosphere bacterial and eukaryotic microbes were able to utilize root exudates. The enrichment of microbial species in the rhizosphere is selective and mostly due to root exudation, which functions as a nutrition source, promoting the growth of these microbes.

The aim of this research was to identify key microorganisms for thermophilic (55 °C) anaerobic digestion of thin stillage derived from hydrolysis and ethanol fermentation of energy cane in a conventional stirred tank reactor with a 10-day hydraulic retention time. Efficient thermophilic anaerobic digestion with a specific methane production of 0.43 Lmethane/gtCOD used/d and biogas containing around 56% methane was accomplished. Due to an overnight temperature perturbation the specific methane production decreased to 0.16 Lmethane/gtCOD used/d. Analysis of the microbial community showed the importance of methanogenic Archaea belonging to Methanosarcina and Methanothermobacter as well as syntrophic Bacteria related to Thermacetogenium, Tepidanaerobacter and Anaerobaculum. This indicates that retention of biomass maintaining syntrophy and methanogenesis more efficiently may be useful for thermophilic anaerobic digestion of thin stillage derived from the production of energy cane ethanol.

OBJECTIVES: Many factors influence the development of the infant microbiota. Based on 16S rDNA sequencing information, the type of feeding, i.e., breast vs formula, has been shown to be the most important factor, with route of delivery having transient effects in the early postpartum period. However, few recent studies have isolated individual strains from human infants or have investigated the functional properties of the infant gut microbes. We used a 2 × 2 experimental design with the goal of determining how diet and delivery interact to shape infant microbiota composition and metabolic activity. METHODS: Fresh fecal samples were collected from 3-month-old infants who were either exclusively breast- (BF) or formula-fed (FF) and cesarean- (CD) or vaginally-delivered (VD). Samples were diluted and plated on two types of media: Gut Microbiota Medium (GMM) or MRS medium. Individual colonies were picked, purified and their 16S rRNA gene sequenced for identification. These isolates were screened for growth on various carbon substrates, including human milk oligosaccharides, prebiotics and volatile fatty acids for 24–36h. Medium supernatant was collected and metabolic profiles assessed by HPLC. RESULTS: More than 150 bacterial strains have been isolated and identified from infant fecal samples to date. Isolates from BF/VD fecal samples were predominantly from the phylum Actinobacteria (dominated with Bifidobacterium) followed by Firmicutes and Proteobacteria. In contrast, isolates from FF/VD or FF/CD infants were primarily from the phylum Firmicutes with only a few Actinobacterial and Proteobacterial isolates. Quantitatively, most isolates represented the genera Bifidobacterium, followed by Enterococcus, and Ruminococcus. On-going phenotypic evaluation of the isolated strains for the growth and utilization of various carbon sources and metabolic profiles will provide insight into species- and strain-specific utilization of common prebiotics in human milk and infant formula. CONCLUSIONS: This collective data will provide novel insights on how the physiological and metabolic function of the infant gut microbiota is influenced by route of delivery and early infant nutrition. FUNDING SOURCES: NIH R01DK107561.

Rhizosphere bacteria have significant contributions to crop health, productivity and carbon sequestration. As maize (Zea mays) is an important economic crop, its rhizosphere bacterial communities have been intensively investigated using various approaches. However, low-resolution profiling methods often make it difficult to understand the complicated rhizosphere bacterial communities and their dynamics. In this study, we analyzed growth-stage related dynamics of bacterial community structures in the rhizosphere of maize using the pyrosequencing method, which revealed an assembly of bacteria enriched in the rhizosphere. Our results revealed that the rhizosphere of maize was preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum was represented by one or two dominating subsets of bacterial groups. Dominant genera enriched in the rhizosphere included Massilia, Burkholderia, Ralstonia, Dyella, Chitinophaga and Sphingobium. Rhizosphere bacterial community structures significantly changed through different growth stages at lower taxonomic ranks (family, genus and OTU levels). Genera Massilia, Flavobacterium, Arenimonas and Ohtaekwangia were relatively abundant at early growth stages, while genera Burkholderia, Ralstonia, Dyella, Chitinophaga, Sphingobium, Bradyrhizobium and Variovorax populations were dominant at later stages. Comparisons of pyrosequencing data collected in Illinois, USA in this study with the available data from Braunschweig, Germany indicated many common bacterial inhabitants but also many differences in the structure of bacterial communities, implying that some site-specific factors, such as soil properties, may play important roles in shaping the structure of rhizosphere bacterial community.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.

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.

Water scarcity is a global problem, and is particularly acute in certain regions like Africa, the Middle East, as well as the western states of America. A breakdown on water usage revealed that 70% of freshwater supplies are used for agricultural irrigation. The use of reclaimed water as an alternative water source for agricultural irrigation would greatly alleviate the demand on freshwater sources. This paradigm shift is gaining momentum in several water scarce countries like Saudi Arabia. However, microbial problems associated with reclaimed water may hinder the use of reclaimed water for agricultural irrigation. Of particular concern is that the occurrence of antibiotic residues in the reclaimed water can select for antibiotic resistance genes among the microbial community. Antibiotic resistance genes can be associated with mobile genetic elements, which in turn allow a promiscuous transfer of resistance traits from one bacterium to another. Together with the pathogens that are present in the reclaimed water, antibiotic resistant bacteria can potentially exchange mobile genetic elements to create the “perfect microbial storm”. Given the significance of this issue, a deeper understanding of the occurrence of antibiotics in reclaimed water, and their potential influence on the selection of resistant microorganisms would be essential. In this review paper, we collated literature over the past two decades to determine the occurrence of antibiotics in municipal wastewater and livestock manure. We then discuss how these antibiotic resistant bacteria may impose a potential microbial risk to the environment and public health, and the knowledge gaps that would have to be addressed in future studies. Overall, the collation of the literature in wastewater treatment and agriculture serves to frame and identify potential concerns with respect to antibiotics, antibiotic resistant bacteria, and antibiotic resistance genes in reclaimed water.

This study aimed to determine if biotic contaminants originating from pig production farms are disseminated into soil and groundwater microbial communities. A spatial and temporal sampling of soil and groundwater in proximity to pig production farms was conducted, and quantitative PCR (Q-PCR) was utilized to determine the abundances of tetracycline resistance genes (i.e., tetQ and tetZ ) and integrase genes (i.e., intI1 and intI2 ). We observed that the abundances of tetZ , tetQ , intI1 , and intI2 in the soils increased at least 6-fold after manure application, and their abundances remained elevated above the background for up to 16 months. Q-PCR further determined total abundances of up to 5.88 × 10 9 copies/ng DNA for tetZ , tetQ , intI1 , and intI2 in some of the groundwater wells that were situated next to the manure lagoon and in the facility well used to supply water for one of the farms. We further utilized 16S rRNA-based pyrosequencing to assess the microbial communities, and our comparative analyses suggest that most of the soil samples collected before and after manure application did not change significantly, sharing a high Bray-Curtis similarity of 78.5%. In contrast, an increase in Bacteroidetes and sulfur-oxidizing bacterial populations was observed in the groundwaters collected from lagoon-associated groundwater wells. Genera associated with opportunistic human and animal pathogens, such as Acinetobacter , Arcobacter , Yersinia , and Coxiella , were detected in some of the manure-treated soils and affected groundwater wells. Feces-associated bacteria such as Streptococcus , Erysipelothrix , and Bacteroides were detected in the manure, soil, and groundwater ecosystems, suggesting a perturbation of the soil and groundwater environments by invader species from pig production activities.

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli , and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k cat values of ∼8,000 and ∼4,500 s −1 , respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.

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.

Metatranscriptomics—gene express profiling via DNA sequencing—is a powerful tool to identify genes that are actively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial community (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhouse gas twenty times more potent than carbon dioxide, and is of general importance for the physiological wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socioeconomically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accurate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-incubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with previously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.

Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.

Many human diets contain arabinoxylan and the ease of genome sequencing coupled with reduced cost have led to unraveling the arsenal of genes utilized by the colonic Bacteroidetes to depolymerize this polysaccharide. The colonic Bacteroidetes with potential to ferment arabinoxylans include Bacteroides intestinalis. In this study, we analyzed the hydrolytic activities of members of a xylan degradation cluster encoded on the genome of Bacteroides intestinalis DSM 17393. Here, it is demonstrated that a cocktail of the xylanolytic enzymes completely hydrolyze arabinoxylans found in human diets. We show that this bacterium and relatives have evolved and secrete a unique bifunctional endoxylanase/arabinofuranosidase in the same polypeptide. The bifunctional enzyme and other secreted enzymes attack the polysaccharides extracellularly to remove the side-chains, exposing the xylan backbone for cleavage to xylo-oligosaccharides and xylose. These end products are transported into the cell where a β-xylosidase cleaves the oligosaccharides to fermentable sugars. While our experiments focused on B. intestinalis, it is likely that the extracellular enzymes also release nutrients to members of the colonic microbial community that practice cross-feeding. The presence of the genes characterized in this study in other colonic Bacteroidetes suggests a conserved strategy for energy acquisition from arabinoxylan, a component of human diets.

A novel halophilic and metal-reducing bacterium,Orenia metallireducensstrain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity withOrenia marismortuibut demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genusOrenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H2as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6 to 9.6), salinity (0.4 to 3.5 M NaCl), and temperature (20 to 60°C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides, such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH), and hematite (α-Fe2O3). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe3(PO4)2] and siderite (FeCO3). The draft genome assembled for strain Z6 is 3.47 Mb in size and contains 3,269 protein-coding genes. Unlike the well-understood iron-reducingShewanellaandGeobacterspecies, this organism lacks thec-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genusOrenia(orderHalanaerobiales) reported to reduce ferric iron minerals and other metal oxides. This microbe expands both the phylogenetic and physiological scopes of iron-reducing microorganisms known to inhabit the deep subsurface and suggests new mechanisms for microbial iron reduction. These distinctions from otherOreniaspp. support the designation of strain Z6 as a new species,Orenia metallireducenssp. nov.IMPORTANCEA novel iron-reducing species,Orenia metallireducenssp. nov., strain Z6, was isolated from groundwater collected from a geological formation located 2.02 km below land surface in the Illinois Basin, USA. Phylogenetic, physiologic, and genomic analyses of strain Z6 found it to have unique properties for iron reducers, including (i) active microbial iron-reducing capacity under broad ranges of temperatures (20 to 60°C), pHs (6 to 9.6), and salinities (0.4 to 3.5 M NaCl), (ii) lack ofc-type cytochromes typically affiliated with iron reduction inGeobacterandShewanellaspecies, and (iii) being the only member of theHalanaerobialescapable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

Ruminococcus albus 8 is a specialist plant cell wall degrading ruminal bacterium capable of utilizing hemicellulose and cellulose. Cellulose degradation requires a suite of enzymes including endoglucanases, exoglucanases and β-glucosidases. The enzymes employed by R. albus 8 in degrading cellulose are yet to be completely elucidated. Through bioinformatic analysis of a draft genome sequence of R. albus 8, seventeen putatively cellulolytic genes were identified. The genes were heterologously expressed in E. coli and purified to near homogeneity. On biochemical analysis with cellulosic substrates, seven of the gene products (Ra0185, Ra0259, Ra0325, Ra0903, Ra1831, Ra2461 and Ra2535) were identified as endoglucanases, releasing predominantly cellobiose and cellotriose. Each of the R. albus 8 endoglucanases, except for Ra0259 and Ra0325, bound to the model crystalline cellulose Avicel, confirming functional carbohydrate binding modules (CBMs). The polypeptides for Ra1831 and Ra2535 were found to contain distantly related homologs of CBM65. Mutational analysis of residues within the CBM65 of Ra1831 identified key residues required for binding. Phylogenetic analysis of the endoglucanases revealed three distinct subfamilies of glycoside hydrolase family 5 (GH5). Our results demonstrate that this fibrolytic bacterium uses diverse GH5 catalytic domains appended with different CBMs, including novel forms of CBM65, to degrade cellulose.