Interspecies cross-feeding orchestrates carbon degradation in the rumen ecosystem.

Because of their agricultural value, there is a great body of research dedicated to understanding the microorganisms responsible for rumen carbon degradation. However, we lack a holistic view of the microbial food web responsible for carbon processing in this ecosystem. Here, we sampled rumen-fistulated moose, allowing access to rumen microbial communities actively degrading woody plant biomass in real time. We resolved 1,193 viral contigs and 77 unique, near-complete microbial metagenome-assembled genomes, many of which lacked previous metabolic insights. Plant-derived metabolites were measured with NMR and carbohydrate microarrays to quantify the carbon nutrient landscape. Network analyses directly linked measured metabolites to expressed proteins from these unique metagenome-assembled genomes, revealing a genome-resolved three-tiered carbohydrate-fuelled trophic system. This provided a glimpse into microbial specialization into functional guilds defined by specific metabolites. To validate our proteomic inferences, the catalytic activity of a polysaccharide utilization locus from a highly connected metabolic hub genome was confirmed using heterologous gene expression. Viral detected proteins and linkages to microbial hosts demonstrated that phage are active controllers of rumen ecosystem function. Our findings elucidate the microbial and viral members, as well as their metabolic interdependencies, that support in situ carbon degradation in the rumen ecosystem.

New roles in hemicellulosic sugar fermentation for the uncultivated Bacteroidetes family BS11.

Ruminants have co-evolved with their gastrointestinal microbial communities that digest plant materials to provide energy for the host. Some arctic and boreal ruminants have already shown to be vulnerable to dietary shifts caused by changing climate, yet we know little about the metabolic capacity of the ruminant microbiome in these animals. Here, we use meta-omics approaches to sample rumen fluid microbial communities from Alaskan moose foraging along a seasonal lignocellulose gradient. Winter diets with increased hemicellulose and lignin strongly enriched for BS11, a Bacteroidetes family lacking cultivated or genomically sampled representatives. We show that BS11 are cosmopolitan host-associated bacteria prevalent in gastrointestinal tracts of ruminants and other mammals. Metagenomic reconstruction yielded the first four BS11 genomes; phylogenetically resolving two genera within this previously taxonomically undefined family. Genome-enabled metabolic analyses uncovered multiple pathways for fermenting hemicellulose monomeric sugars to short-chain fatty acids (SCFA), metabolites vital for ruminant energy. Active hemicellulosic sugar fermentation and SCFA production was validated by shotgun proteomics and rumen metabolites, illuminating the role BS11 have in carbon transformations within the rumen. Our results also highlight the currently unknown metabolic potential residing in the rumen that may be vital for sustaining host energy in response to a changing vegetative environment.

Summer dietary nitrogen availability as a potential bottom-up constraint on moose in south-central Alaska.

Recent studies suggest that the growth and fecundity of northern ungulates may be coupled to their summer nutrition. Here, we compare summer dietary nitrogen availability of the five major browse plants (comprising approximately 79% of the diet) of moose (Alces alces) in Denali National Park and Nelchina Basin, Alaska, USA. In recent years the productivity of Denali moose has been significantly higher than that of Nelchina moose, prompting this comparison. We examined the phenological progression of leaf nitrogen concentration, tannin-protein precipitation capacity, and digestible protein over three summers in both regions. We then modeled the potential nutritional consequences for a cow moose consuming representative diets on each range, predicting both net protein intake (NPI) and lean body mass accumulation each year. We found that leaf nitrogen and digestible protein decreased, while tannin-protein precipitation capacity increased throughout the summer for all forages. There was 23% more digestible protein in Denali leaves than Nelchina leaves on average, and this difference was significant in all three years. Tannins accounted for a large (mean = 46%) reduction in protein availability, suggesting a key role of these secondary compounds in the nitrogen balance of moose in these regions. Finally, our NPI model predicted that Denali cows were in positive protein balance 17 days longer than Nelchina cows and accumulated 18 kg more lean body mass over the summer, on average. We conclude that summer dietary nitrogen availability may act as a nutritional constraint on moose and suggest that more emphasis be placed on elucidating its role in population dynamics and conservation of northern ungulates.

A modified method for determining tannin-protein precipitation capacity using accelerated solvent extraction (ASE) and microplate gel filtration.

The protein precipitation assay used by Robbins et al., (1987) Ecology 68:98-107 has been shown to predict successfully the reduction in protein availability to some ruminants due to tannins. The procedure, however, is expensive and laborious, which limits its utility, especially for quantitative ecological or nutritional applications where large numbers of assays may be required. We have modified the method to decrease its cost and increase laboratory efficiency by: (1) automating the extraction by using Accelerated Solvent Extraction (ASE); and (2) by scaling and automating the precipitation reaction, chromatography, and spectrometry with microplate gel filtration and an automated UV-VIS microplate spectrometer. ASE extraction is shown to be as effective at extracting tannins as the hot methanol technique. Additionally, the microplate assay is sensitive and precise. We show that the results from the new technique correspond in a nearly 1:1 relationship to the results of the previous technique. Hence, this method could reliably replace the older method with no loss in relevance to herbivore protein digestion. Moreover, the ASE extraction technique should be applicable to other tannin-protein precipitation assays and possibly other phenolic assays.

Adaptation of ruminants to browse and grass diets: are anatomical-based browser-grazer interpretations valid?

As a result of pioneering work of Hofmann (1973, 1989), nutritional ecologists classify ruminants into three feeding-type categories: browsers ("concentrate" feeders), grazers, and intermediate or mixed feeders. Hofmann proposed that these feeding types result from evolutionary adaptations in the anatomy of the digestive system and that one consequence is shorter retention of the digesta in the rumen of browsers, and thus a decreased efficiency of fiber digestion relative to that of grazers. We examined the hypotheses that (1) fiber digestion of browsers is lower than that of grazers, (2) salivary gland size is larger in all browsers than in grazers, (3) the browser's larger salivary glands produce larger volumes of thin serous saliva than those of grazers, and (4) thus, browsers have higher liquid passage rates than do grazers. We found that the extent of fiber digestion is not significantly different between browsers and grazers, although fiber digestion is positively related to herbivore size. In general, salivary gland size is approximately 4 times larger in browsers than grazers, but some browsers (e.g., greater kudu) have small, grazer-sized salivary glands. Resting (non-feeding or ruminating) saliva flow rates of mule deer (browser) and domestic sheep and cattle (grazers) were not significantly different from each other. Finally, ruminal liquid flow rates were not different between feeding types. We conclude that many of Hofmann's nutritional and physiological interpretations of anatomical differences amongst ruminants are not supportable.