Thermophilic Degradation of Hemicellulose, a Critical Feedstock in the Production of Bioenergy and Other Value-Added Products.

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.

Assessment of microbial diversity associated with CH4 emission from sugarcane vinasse storage and transportation systems.

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.

Generation of an E. coli platform strain for improved sucrose utilization using adaptive laboratory evolution.

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.

Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity.

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.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus.

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 ß-l-arabinopyranosidase (CpAbp27A), and two GH127 ß-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-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 ß-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. polysaccharolyticusIMPORTANCE 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 ß-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.

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

Orenia metallireducens sp. nov. Strain Z6, a Novel Metal-Reducing Member of the Phylum Firmicutes from the Deep Subsurface.

A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain 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 with Orenia marismortui but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H2 as 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-reducing Shewanella and Geobacter species, this organism lacks the c-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genus Orenia (order Halanaerobiales) 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 other Orenia spp. support the designation of strain Z6 as a new species, Orenia metallireducens sp. nov. IMPORTANCE: A novel iron-reducing species, Orenia metallireducens sp. 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 of c-type cytochromes typically affiliated with iron reduction in Geobacter and Shewanella species, and (iii) being the only member of the Halanaerobiales capable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17.

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 ß-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.

Metagenomic-based study of the phylogenetic and functional gene diversity in Galápagos land and marine iguanas.

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

The gut microbiota appears to compensate for seasonal diet variation in the wild black howler monkey (Alouatta pigra).

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.

Hydrogen formation and its regulation in Ruminococcus albus: involvement of an electron-bifurcating [FeFe]-hydrogenase, of a non-electron-bifurcating [FeFe]-hydrogenase, and of a putative hydrogen-sensing [FeFe]-hydrogenase.

Ruminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO2, and 2.6 H2 when growing in batch culture and to 2 acetate, 2 CO2, and 4 H2 when growing in continuous culture in syntrophic association with H2-consuming microorganisms that keep the H2 partial pressure low. The organism uses NAD(+) and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO2, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H2. Of all the enzymes involved, only the enzyme catalyzing the formation of H2 from NADH remained unknown. Here, we report that R. albus 7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]-hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H2 from NADH to the exergonic formation of H2 from reduced ferredoxin. Interestingly, hydA2 is adjacent to the hydS gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that hydS and hydA2 are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when R. albus grows at high H2 partial pressures, HydS could be a H2-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis.

Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir.

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.

Evolution of Salmonella enterica virulence via point mutations in the fimbrial adhesin.

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella.

Nitrogen utilization and metabolism in Ruminococcus albus 8.

The model rumen Firmicutes organism Ruminococcus albus 8 was grown using ammonia, urea, or peptides as the sole nitrogen source; growth was not observed with amino acids as the sole nitrogen source. Growth of R. albus 8 on ammonia and urea showed the same growth rate (0.08 h(-1)) and similar maximum cell densities (for ammonia, the optical density at 600 nm [OD600] was 1.01; and for urea, the OD600 was 0.99); however, growth on peptides resulted in a nearly identical growth rate (0.09 h(-1)) and a lower maximum cell density (OD600 = 0.58). To identify differences in gene expression and enzyme activities, the transcript abundances of 10 different genes involved in nitrogen metabolism and specific enzyme activities were analyzed by harvesting mRNA and crude protein from cells at the mid- and late exponential phases of growth on the different N sources. Transcript abundances and enzyme activities varied according to nitrogen source, ammonia concentration, and growth phase. Growth of R. albus 8 on ammonia and urea was similar, with the only observed difference being an increase in urease transcript abundance and enzyme activity in urea-grown cultures. Growth of R. albus 8 on peptides showed a different nitrogen metabolism pattern, with higher gene transcript abundance levels of gdhA, glnA, gltB, amtB, glnK, and ureC, as well as higher activities of glutamate dehydrogenase and urease. These results demonstrate that ammonia, urea, and peptides can all serve as nitrogen sources for R. albus and that nitrogen metabolism genes and enzyme activities of R. albus 8 are regulated by nitrogen source and the level of ammonia in the growth medium.

Two new xylanases with different substrate specificities from the human gut bacterium Bacteroides intestinalis DSM 17393.

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit ß-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

The role of gut microbes in satisfying the nutritional demands of adult and juvenile wild, black howler monkeys (Alouatta pigra).

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.

Dietary supplementation of usnic acid, an antimicrobial compound in lichens, does not affect rumen bacterial diversity or density in reindeer.

Reindeer (Rangifer tarandus tarandus) may include large proportions of lichens in their winter diet. These dietary lichens are rich in phenolic secondary compounds, the most well-known being the antimicrobial usnic acid. Previous studies have shown that reindeer host rumen bacteria resistant to usnic acid and that usnic acid is quickly detoxified in their rumen. In the present study, reindeer (n = 3) were sampled before, during, and after usnic acid supplementation to determine the effect on their rumen microbial ecology. Ad libitum intake of usnic acid averaged up to 278 mg/kg body mass. Population densities of rumen bacteria and methanogenic archaea determined by real-time PCR, ranged from 1.36 × 10(9) to 11.8 × 10(9) and 9.0 × 10(5) to 1.35 × 10(8) cells/g wet weight, respectively, and the two populations did not change significantly during usnic acid supplementation (repeated measures ANOVA) or vary significantly between the rumen liquid and particle fraction (paired t test). Rumen bacterial community structure determined by denaturing gradient gel electrophoresis did not change in response to intake of usnic acid. Firmicutes (38.7 %) and Bacteriodetes (27.4 %) were prevalent among the 16S rRNA gene sequences (n = 62) from the DGGE gels, but representatives of the phyla Verrucomicrobia (14.5 %) and Proteobacteria (1.6 %) were also detected. Rapid detoxification of the usnic acid or resistance to usnic acid may explain why the diversity of the dominant bacterial populations and the bacterial density in the reindeer rumen does not change during usnic acid supplementation.

- Invited Review - Hydrogen production and hydrogen utilization in the rumen: key to mitigating enteric methane production.

Molecular hydrogen (H2) and formate (HCOO-) are metabolic end products of many primary fermenters in the rumen ecosystem. 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 rumen, 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 possibly homoacetogens play a key role in maintaining the metabolic efficiency of primary fermenters. There is increasing interest in identifying approaches to manipulate the rumen ecosystem 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 utilization could be a significant starting point for the development of successful interventions aimed at redirecting electron flow and reducing methane emissions. We conclude by discussing in brief ruminant methane mitigation approaches as a model to help understand the fate of H2 and formate in the rumen ecosystem.

Organ growth and fermentation profiles of broilers differing in body growth rate.

This study sought to determine the relationship among broiler performance, organ development, and indicators of microbiota colonization. A total of 1,200 two-day-old male Ross 308 broiler chicks, divided among 3 cohorts of equal size, were housed in battery cages, and allotted based on body weight. On study d 11, birds were weighed, and birds with BW gain within the 10th and 90th percentiles were assigned to the Slow and Fast groups, respectively. Birds (n = 30 for each group) selected on d 11 were provided water and a corn-soybean meal-based diet ad libitum while maintained individually through study d 25 (i.e., a 14-d growth period). Parameters regarding growth performance, organ and intestine weights and lengths, and intestinal volatile fatty acid concentrations were measured. All data were analyzed by one-way ANOVA using the Mixed procedure of SAS. Fast birds exhibited greater (P < 0.001) BW gain and feed intake than slow birds, but feed conversion ratio (FCR) did not differ (P = 0.19). Additionally, Slow birds had higher (P < 0.05) relative weights (% of BW) for nearly all organs on d 11 and 25, most notably the gizzard, proventriculus, pancreas, and liver. Conversely, intestinal sections were longer (P < 0.05) in the Fast birds. Measurement of gut histomorphology did not show any notable differences between growth rate groups in terms of villi height, crypt depth, or their ratio for either time-point (P > 0.05). In terms of volatile fatty acid concentrations of luminal contents, acetate concentrations were 10.2% higher (P < 0.001) in the ileum of the Slow birds compared with Fast birds on d 25. Overall, the findings suggest that total BW gain is influenced by the development of metabolically active organs, as supported by lower weight gain in Slow birds with relatively larger organ weights and shorter intestinal lengths than their Fast counterparts. The general lack of differences in fermentation end-product concentrations in luminal contents does not rule out influence of the microbiota on growth rate of broilers, which warrants further investigation.

Biochemical and structural insights into xylan utilization by the thermophilic bacterium Caldanaerobius polysaccharolyticus.

Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.