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Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/β hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome., (© 2024. His Majesty the King in Right of Canada as represented by the Minister of Agriculture and Agri-Food, and Liam Worrel.)

The individual and combined effects of 3-nitrooxypropanol (3-NOP) and canola oil (OIL) supplementation on enteric methane (CH4) and hydrogen (H2) emissions, rumen fermentation and biohydrogenation, and total tract nutrient digestibility were investigated in beef cattle. Eight beef heifers (mean body weight ± SD, 732 ± 43 kg) with ruminal fistulas were used in a replicated 4 × 4 Latin square with a 2 (with and without 3-NOP) × 2 (with and without OIL) arrangement of treatments and 28-d periods (13 d adaption and 15 d measurements). The four treatments were: control (no 3-NOP, no OIL), 3-NOP (200 mg/kg dry matter [DM]), OIL (50 g/kg DM), and 3-NOP (200 mg/kg DM) plus OIL (50 g/kg DM). Animals were fed restrictively (7.6 kg DM/d) a basal diet of 900 g/kg DM barley silage and 100 g/kg DM supplement. 3-NOP and OIL decreased (P < 0.01) CH4 yield (g/kg DM intake) by 31.6% and 27.4%, respectively, with no 3-NOP × OIL interaction (P = 0.85). Feeding 3-NOP plus OIL decreased CH4 yield by 51% compared with control. There was a 3-NOP × OIL interaction (P = 0.02) for H2 yield (g/kg DM intake); the increase in H2 yield (P < 0.01) due to 3-NOP was less when it was combined with OIL. There were 3-NOP × OIL interactions for molar percentages of acetate and propionate (P < 0.01); individually, 3-NOP and OIL decreased acetate and increased propionate percentages with no further effect when supplemented together. 3-NOP slightly increased crude protein (P = 0.02) and starch (P = 0.01) digestibilities, while OIL decreased the digestibilities of DM (P < 0.01) and neutral detergent fiber (P < 0.01) with no interactions (P = 0.15 and 0.10, respectively). 3-NOP and OIL increased (P = 0.04 and P < 0.01, respectively) saturated fatty acid concentration in rumen fluid, with no interaction effect. Interactions for ruminal trans-monounsaturated fatty acids (t-MUFA) concentration and percentage were observed (P = 0.02 and P < 0.01); 3-NOP had no effect on t-MUFA concentration and percentage, while OIL increased the concentration (P < 0.01) and percentage (P < 0.01) of t-MUFA but to a lesser extent when combined with 3-NOP. In conclusion, the CH4-mitigating effects of 3-NOP and OIL were independent and incremental. Supplementing ruminant diets with a combination of 3-NOP and OIL may help mitigate CH4 emissions, but the decrease in total tract digestibility due to OIL may decrease animal performance and needs further investigation., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2021.)

3-Nitrooxypropanol (3-NOP) is an investigational compound that acts as an enzyme inhibitor to decrease ruminal methanogenesis. We hypothesized that when feeding 3-NOP to cattle fed a high-forage diet, H 2 would accumulate in the rumen, which could suppress microbial colonization of feed particles and fiber degradation. Therefore, the study investigated the effects of supplementing a high-forage diet with 3-NOP on ruminal fiber degradability and microbial colonization of feed particles using the in situ technique. Eight ruminally cannulated beef cattle were allocated to 2 groups (4 cattle/group) in a crossover design with 2 periods and 2 dietary treatments. The treatments were control (basal diet) and 3-NOP (basal diet supplemented with 3-NOP, 150 mg/kg of dry matter). The basal diet consisted of 45% barley silage, 45% chopped grass hay, and 10% concentrate (dry matter basis). Samples of dried, ground barley silage and grass hay were incubated in the rumen of each animal for 0, 4, 12, 24, 36, 48, 96, 120, 216, and 288 h to determine neutral detergent fiber (NDF) degradation kinetics. An additional 2 bags were incubated for 4 and 48 h to evaluate the bacterial community attached to the incubated forages. Dietary supplementation of 3-NOP decreased (-53%) the dissolved methane concentration and increased (+780%) the dissolved H 2 concentration in ruminal fluid, but did not substantially alter in situ NDF degradation. The addition of 3-NOP resulted in a decrease in the α-diversity of the microbial community with colonizing communities showing reduced numbers of amplicon sequence variants and phylogenetic diversity compared with control diets. Principal coordinate analysis plots indicated that forages incubated in animals fed 3-NOP resulted in highly specific changes to targeted microbes compared with control diets based on unweighted analysis (considering only absence and presence of taxa), but did not alter the overall composition of the colonizing community based on weighted UniFrac distances; unchanged relative abundances of major taxa included phyla Bacteroidetes, Firmicutes, and Fibrobacteres. The effect of 3-NOP on colonizing methanogenic microbes differed depending upon the forage incubated, as abundance of genus Methanobrevibacter was decreased for barley silage but not for grass hay. In conclusion, 3-NOP supplementation of a high-forage diet decreased ruminal methanogenesis and increased dissolved H 2 concentration, but had no negative effects on ruminal fiber degradation and only minor effects on relative abundances of the major taxa of bacteria adhered to forage substrates incubated in the rumen., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

This study examines the colonization of barley straw (BS) and corn stover (CS) by rumen bacteria and how this is impacted by ammonia fiber expansion (AFEX) pre-treatment. A total of four ruminally cannulated beef heifers were used to investigate in situ microbial colonization in a factorial design with two crop residues, pre-treated with or without AFEX. Crop residues were incubated in the rumen for 0, 2, 4, 8 and 48 h and the colonizing profile was determined using 16 s rRNA gene sequencing. The surface colonizing community clustered based on incubation time and pre-treatment. Fibrobacter, unclassified Bacteroidales, and unclassified Ruminococcaceae were enriched during late stages of colonization. Prevotella and unclassified Lachnospiraceae were enriched in the early stages of colonization. The microbial community colonizing BS-AFEX and CS was less diverse than the community colonizing BS and CS-AFEX. Prevotella, Coprococcus and Clostridium were enriched in both AFEX crop residues, while untreated crop residues were enriched with Methanobrevibacter. Several pathways associated with simple carbohydrate metabolism were enriched in the primary colonizing community of AFEX crop residues. This study suggests that AFEX improves the degradability of crop residues by increasing the accessibility of polysaccharides that can be metabolized by the dominant taxa responsible for primary colonization., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-food Canada, 2020.)

Ruminants play an important role in food security, but there is a growing concern about the impact of cattle on the environment, particularly regarding greenhouse gas emissions. The objective of this study was to examine the effect of humic substances (HS) on rumen fermentation, nutrient digestibility, methane (CH4) emissions, and the rumen microbiome of beef heifers fed a barley silage-based diet. The experiment was designed as a replicated 4 × 4 Latin square using 8 ruminally cannulated Angus × Hereford heifers (758 ± 40.7 kg initial BW). Heifers were offered a basal diet consisting of 60% barley silage and 40% concentrate (DM basis) with either 0- (control), 100-, 200- or 300-mg granulated HS/kg BW. Each period was 28 d with 14 d of adaptation. Rumen samples were taken on day 15 at 0, 3, 6, and 12 h postfeeding. Total urine and feces were collected from days 18 to 22. Blood samples were taken on day 22 at 0 and 6 h postfeeding. Between days 26 and 28, heifers were placed in open-circuit respiratory chambers to measure CH4. Ruminal pH was recorded continuously during the periods of CH4 measurement using indwelling pH loggers. Intake was similar (P = 0.47) across treatments. Concentration of ammonia-N and counts of rumen protozoa responded quadratically (P = 0.03), where both increased at H100 and then decreased for the H300 treatments. Apparent total tract digestibility of CP (P = 0.04) was linearly increased by HS and total N retention (g/d, % N intake, g/kg BW0.75) was improved (P = 0.04) for HS when compared with the control. There was no effect of HS on CH4 production (g/d; P = 0.83); however, HS decreased the relative abundance of Proteobacteria (P = 0.04) and increased the relative abundance of Synergistetes (P = 0.01) and Euryarchaeota (P = 0.04). Results suggest that HS included at up to 300 mg/kg BW may improve N retention and CP digestibility, but there was no impact on CH4 production., (© Crown copyright 2018.)

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes ( nf2152 , nf2215 , nf2523 , and pr2455 ) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, β1,2-arabinobiose (Ara 2 ) from sugar beet arabinan (SBA), and β1,2-Ara 2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both β-1,2-Ara 2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Future growth in demand for meat and milk, and the socioeconomic and environmental challenges that farmers face, represent a "grand challenge for humanity". Improving the digestibility of crop residues such as straw could enhance the sustainability of ruminant production systems. Here, we investigated if transfer of rumen contents from bison to cattle could alter the rumen microbiome and enhance total tract digestibility of a barley straw-based diet. Beef heifers were adapted to the diet for 28 days prior to the experiment. After 46 days, ~70 percent of rumen contents were removed from each heifer and replaced with mixed rumen contents collected immediately after slaughter from 32 bison. This procedure was repeated 14 days later. Intake, chewing activity, total tract digestibility, ruminal passage rate, ruminal fermentation, and the bacterial and protozoal communities were examined before the first and after the second transfer. Overall, inoculation with bison rumen contents successfully altered the cattle rumen microbiome and metabolism, and increased protein digestibility and nitrogen retention, but did not alter fiber digestibility.

Interest in the bacteria responsible for the breakdown of lignocellulosic feedstuffs within the rumen has increased due to their potential utility in industrial applications. To date, most studies have focused on bacteria from domesticated ruminants. We have expanded the knowledge of the microbial ecology of ruminants by examining the bacterial populations found in the rumen of non-domesticated ruminants found in Canada. Next-generation sequencing of 16S rDNA was employed to characterize the liquid and solid-associated bacterial communities in the rumen of elk (Cervus canadensis), and white tailed deer (Odocoileus virginianus). Despite variability in the microbial populations between animals, principle component and weighted UniFrac analysis indicated that bacterial communities in the rumen of elk and white tail deer are distinct. Populations clustered according to individual host animal and not the association with liquid or solid phase of the rumen contents. In all instances, Bacteroidetes and Firmicutes were the dominant bacterial phyla, although the relative abundance of these differed among ruminant species and between phases of rumen digesta, respectively. In the elk samples Bacteroidetes were more predominant in the liquid phase whereas Firmicutes was the most prevalent phyla in the solid digesta (P = 1×10(-5)). There were also statistically significant differences in the abundance of OTUs classified as Fibrobacteres (P = 5×10(-3)) and Spirochaetes (P = 3×10(-4)) in the solid digesta of the elk samples. We identified a number of OTUs that were classified as phylotypes not previously observed in the rumen environment. Our results suggest that although the bacterial diversity in wild North American ruminants shows overall similarities to domesticated ruminants, we observed a number of OTUs not previously described. Previous studies primarily focusing on domesticated ruminants do not fully represent the microbial diversity of the rumen and studies focusing on non-domesticated ruminants should be expanded.

Background: Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the ellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow., Results: Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum., Conclusion: The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.