Diet Influences Early Microbiota Development in Dairy Calves without Long-Term Impacts on Milk Production.

Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and affect the production of agriculturally relevant products, including milk and meat. Despite this link, interventions to alter the adult microbiota to improve production have proven ineffective, as established microbial communities are resilient to change. In contrast, developing communities in young animals may be more easily altered but are less well studied. Here, we measured the GIT-associated microbiota of 45 Holstein dairy cows from 2 weeks to the first lactation cycle, using Illumina amplicon sequencing of bacterial (16S rRNA V4), archaeal (16S rRNA V6 to V8), and fungal (internal transcribed region 1 [ITS1]) communities. Fecal and ruminal microbiota of cows raised on calf starter grains and/or corn silage were correlated to lifetime growth as well as milk production during the first lactation cycle, in order to determine whether early-life diets have long-term impacts. Significant diet-associated differences in total microbial communities and specific taxa were observed by weaning (8 weeks), but all animals reached an adult-like composition between weaning and 1 year. While some calf-diet-driven differences were apparent in the microbiota of adult cows, these dissimilarities did not correlate with animal growth or milk production. This finding suggests that initial microbial community establishment is affected by early-life diet but postweaning factors have a greater influence on adult communities and production outcomes.IMPORTANCE The gut microbiota is essential for the survival of many organisms, including ruminants that rely on microorganisms for nutrient acquisition from dietary inputs for the production of products such as milk and meat. While alteration of the adult ruminant microbiota to improve production is possible, changes are often unstable and fail to persist. In contrast, the early-life microbiota may be more amenable to sustained modification. However, few studies have determined the impact of early-life interventions on downstream production. Here, we investigated the impact of agriculturally relevant calf diets, including calf starter and corn silage, on gut microbial communities, growth, and production through the first lactation cycle. Thus, this work serves to further our understanding of early-life microbiota acquisition, as well as informing future practices in livestock management.

Fermentation of model hemicelluloses by Prevotella strains and Butyrivibrio fibrisolvens in pure culture and in ruminal enrichment cultures.

Hemicelluloses are major components of plant biomass, but their fermentation in the rumens of cattle and other ruminants is poorly understood. We compared four species of the ruminally dominant genus Prevotella and the well-known hemicellulose utilizer, Butyrivibrio fibrisolvens, with respect to degradation of several isolated hemicelluloses (xylans, glucomannan, and xyloglucan). We also performed Illumina sequencing of the V3/V4 region of 16S rRNA genes to determine the relative proportions of Prevotella and Butyrivibrio in hemicellulose-fed enrichment cultures inoculated from ruminal contents of dairy cattle fed a total mixed ration (TMR) rich in hemicelluloses. Results confirmed the xylan fermentation and butyrate production abilities of B. fibrisolvens. Despite their reputation as generalist fermenters, the Prevotella strains poorly fermented these hemicelluloses but exhibited dramatic differences in fermentation end products. Prevotella was much less abundant in mixed bacterial enrichment cultures fed the same TMR than in the ruminal inoculum, yet Prevotella was again the most abundant genus in enrichment cultures fed xylans. By contrast, glucomannan fermentations were dominated by Streptococcus sp. Genera known for hemicellulose degradation (Butyrivibrio, Ruminococcus, and Fibrobacter) were not significantly enriched on these hemicelluloses. Substantial differences in fermentation end product distribution from the different hemicelluloses were observed, which would likely affect nutrient partitioning in the host animal. Differences in community composition between in vitro hemicellulose enrichments and inoculum samples emerged at every phylogenetic level, suggesting that in vitro conditions provide unique selective pressures on the bacterial community and also that ruminal bacteria exhibit specialization with respect to hemicellulose utilization.

Transient changes in milk production efficiency and bacterial community composition resulting from near-total exchange of ruminal contents between high- and low-efficiency Holstein cows.

The objectives of this study were to determine if milk production efficiency (MPE) is altered by near-total exchange of ruminal contents between high- (HE) and low-MPE (LE) cows and to characterize ruminal bacterial community composition (BCC) before exchange and over time postexchange. Three pairs of ruminally cannulated, third-lactation cows were selected whose MPE (energy-corrected milk per unit of dry matter intake) differed over their first 2 lactations. Approximately 95% of ruminal contents were exchanged between cows within each pair. Ruminal pH and volatile fatty acid (VFA) profiles, along with BCC (characterized by sequencing of the variable 4 region of 16S rRNA genes), were assessed just before feeding on d -8, -7, -5, -4, -1, 1, 2, 3, 7, 10, 14, 21, 28, 35, 42, and 56, relative to the exchange date. High-MPE cows had higher total ruminal VFA concentrations, higher molar percentages of propionate and valerate, and lower molar percentages of acetate and butyrate than did LE cows, and re-established these differences 1 d after contents exchange. Across all LE cows, MPE increased during 7 d postexchange but declined thereafter. Two of the 3 HE cows displayed decreases in MPE following introduction of the ruminal contents from the corresponding LE cow, but MPE increased in the third HE cow, which was determined to be an outlier. For all 6 cows, both liquid- and solids-associated BCC differed between individuals within a pair before contents exchange. Upon exchange, BCC of both phases in all 3 pairs was more similar to that of the donor inoculum than to preexchange host BCC. For 5 of 6 cows, the solids-associated community returned within 10 d to more resemble the preexchange community of that host than that of the donor community. Individual variability before the exchange was greater in liquids than in solids, as was the variability in response of bacterial communities to the exchange. Individual cows varied in their response, but generally moved toward re-establishment of their preexchange communities by 10 d after contents exchange. By contrast, ruminal pH and VFA profiles returned to preexchange levels within 1 d. Despite the small number of cows studied, the data suggest an apparent role for the ruminal bacterial community as a determinant of MPE.

Diet specialization selects for an unusual and simplified gut microbiota in two- and three-toed sloths.

Symbiotic microbial communities are critical to the function and survival of animals. This relationship is obligatory for herbivores that engage gut microorganisms for the conversion of dietary plant materials into nutrients such as short-chain organic acids (SCOAs). The constraint on body size imposed by their arboreal lifestyle is thought to make this symbiosis especially important for sloths. Here, we use next-generation sequencing to identify the bacteria present in the fore and distal guts of wild two- and three-toed sloths, and correlate these communities with both diet and SCOAs. We show that, unlike other mammalian herbivores, sloth gut communities are dominated by the bacterial phyla Proteobacteria and Firmicutes. Specifically, three-toed sloths possess a highly conserved, low-diversity foregut community with a highly abundant Neisseria species associated with foregut lactate. In contrast, two-toed sloths have a more variable and diverse foregut microbiota correlated with a variety of SCOAs. These differences support the hypothesis that feeding behaviour selects for specific gut bacterial communities, as three-toed sloths subsist primarily on Cecropia tree leaves while two-toed sloths have a more generalist diet. The less diverse diet and gut microbiota of three-toed sloths may render them more susceptible to habitat loss and other diet-altering conditions.

Impacts of ruminal microorganisms on the production of fuels: how can we intercede from the outside?

The ruminal microbiome rapidly converts plant biomass to short-chain fatty acids (SCFA) that nourish the ruminant animal host. Because of its high species diversity, functional redundancy, and ease of extraruminal cultivation, this mixed microbial community is a particularly accomplished practitioner of the carboxylate platform for producing fuels and chemical precursors. Unlike reactor microbiomes derived from anaerobic digesters or sediments, the ruminal community naturally produces high concentrations of SCFA, with only modest methane production owing to the absence of both proton-reducing acetogens and aceticlastic methanogens. The extraruminal fermentation can be improved by addition of ethanol or lactate product streams, particularly in concert with reverse ß-oxidizing bacteria (e.g., Clostridium kluyveri or Megasphaera elsdenii) that facilitate production of valeric and caproic acids. Application of fundamental principles of thermodynamics allows identification of optimal conditions for SCFA chain elongation, as well as discovery of novel synthetic capabilities (e.g., medium-chain alcohol and alkane production) by this mixed culture system.

Ruminal Bacterial Community Composition in Dairy Cows Is Dynamic over the Course of Two Lactations and Correlates with Feed Efficiency.

Fourteen Holstein cows of similar ages were monitored through their first two lactation cycles, during which ruminal solids and liquids, milk samples, production data, and feed consumption data were collected for each cow during early (76 to 82 days in milk [DIM]), middle (151 to 157 DIM), and late (251 to 257 DIM) lactation periods. The bacterial community of each ruminal sample was determined by sequencing the region from V6 to V8 of the 16S rRNA gene using 454 pyrosequencing. Gross feed efficiency (GFE) for each cow was calculated by dividing her energy-corrected milk by dry matter intake (ECM/DMI) for each period of both lactation cycles. Four pairs of cows were identified that differed in milk production efficiency, as defined by residual feed intake (RFI), at the same level of ECM production. The most abundant phyla detected for all cows were Bacteroidetes (49.42%), Firmicutes (39.32%), Proteobacteria (5.67%), and Tenericutes (2.17%), and the most abundant genera included Prevotella (40.15%), Butyrivibrio (2.38%), Ruminococcus (2.35%), Coprococcus (2.29%), and Succiniclasticum (2.28%). The bacterial microbiota between the first and second lactation cycles were highly similar, but with a significant correlation between total community composition by ruminal phase and specific bacteria whose relative sequence abundances displayed significant positive or negative correlation with GFE or RFI. These data suggest that the ruminal bacterial community is dynamic in terms of membership and diversity and that specific members are associated with high and low milk production efficiency over two lactation cycles.

Unique aspects of fiber degradation by the ruminal ethanologen Ruminococcus albus 7 revealed by physiological and transcriptomic analysis.

BACKGROUND: Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. Here, we used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. RESULTS: A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. CONCLUSIONS: Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.

A syndrome of mutualism reinforces the lifestyle of a sloth.

Arboreal herbivory is rare among mammals. The few species with this lifestyle possess unique adaptions to overcome size-related constraints on nutritional energetics. Sloths are folivores that spend most of their time resting or eating in the forest canopy. A three-toed sloth will, however, descend its tree weekly to defecate, which is risky, energetically costly and, until now, inexplicable. We hypothesized that this behaviour sustains an ecosystem in the fur of sloths, which confers cryptic nutritional benefits to sloths. We found that the more specialized three-toed sloths harboured more phoretic moths, greater concentrations of inorganic nitrogen and higher algal biomass than the generalist two-toed sloths. Moth density was positively related to inorganic nitrogen concentration and algal biomass in the fur. We discovered that sloths consumed algae from their fur, which was highly digestible and lipid-rich. By descending a tree to defecate, sloths transport moths to their oviposition sites in sloth dung, which facilitates moth colonization of sloth fur. Moths are portals for nutrients, increasing nitrogen levels in sloth fur, which fuels algal growth. Sloths consume these algae-gardens, presumably to augment their limited diet. These linked mutualisms between moths, sloths and algae appear to aid the sloth in overcoming a highly constrained lifestyle.

Megasphaera elsdenii: Its Role in Ruminant Nutrition and Its Potential Industrial Application for Organic Acid Biosynthesis.

The Gram-negative, strictly anaerobic bacterium Megasphaera elsdenii was first isolated from the rumen in 1953 and is common in the mammalian gastrointestinal tract. Its ability to use either lactate or glucose as its major energy sources for growth has been well documented, although it can also ferment amino acids into ammonia and branched-chain fatty acids, which are growth factors for other bacteria. The ruminal abundance of M. elsdenii usually increases in animals fed grain-based diets due to its ability to use lactate (the product of rapid ruminal sugar fermentation), especially at a low ruminal pH (<5.5). M. elsdenii has been proposed as a potential dietary probiotic to prevent ruminal acidosis in feedlot cattle and high-producing dairy cows. However, this bacterium has also been associated with milk fat depression (MFD) in dairy cows, although proving a causative role has remained elusive. This review summarizes the unique physiology of this intriguing bacterium and its functional role in the ruminal community as well as its role in the health and productivity of the host animal. In addition to its effects in the rumen, the ability of M. elsdenii to produce C2-C7 carboxylic acids-potential precursors for industrial fuel and chemical production-is examined.

Changes in rumen bacterial community composition in steers in response to dietary nitrate.

The effect of dietary nitrate supplementation on rumen bacterial community composition was examined in beef steers fed either a nitrate-N diet or urea-N diet. An automated method of ribosomal intergenic spacer analysis was applied to solid and liquid fractions of ruminal contents to allow comparison of bacterial communities. Supplemental N source affected relative population size of four amplicon lengths (ALs) in the liquid fraction and three ALs in the solid fraction. Five ALs were more prevalent after adaptation to nitrate. Correspondence analysis indicated that feeding the steers the nitrate-N diet versus urea-N diet changed the bacterial community composition in the liquid but not in the solid fraction. This led to an investigation of the relative sizes of potential nitrate-reducing populations. Mannheimia succiniciproducens, Veillonella parvula, and Campylobacter fetus were obtained from nitrate enrichment culture and quantified by real-time PCR based on 16S rRNA sequence. Nitrate supplementation increased the percentage of C. fetus in the liquid and solid phases, and in solid phase, the percentage of M. succiniciproducens increased. No change in species prevalence was observed for V. parvula. However, even after adaptation to dietary nitrate, the relative population sizes for all three putative nitrate-reducing species were very low (<0.06 % of 16S rRNA gene copy number). The liquid-associated bacterial community composition changed due to nitrate supplementation, and at least part of this change reflects an increase in the species prevalence of C. fetus, a species which is not typically regarded as a ruminal inhabitant.

An insect herbivore microbiome with high plant biomass-degrading capacity.

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.

Isolation, characterization, and quantification of Clostridium kluyveri from the bovine rumen.

A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium containing ethanol as an electron donor and acetate and succinate (common products of rumen fermentation) as electron acceptors. The isolate displayed a narrow substrate range but wide temperature and pH ranges atypical of ruminal bacteria and a maximum specific growth rate near the typical liquid dilution rate of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread among bovine ruminal samples but was present at only very low levels (0.00002% to 0.0002% of bacterial 16S rRNA gene copy number). However, the species was present in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne silage (but not maize silage) that comprised much of the cows' diet. While C. kluyveri may account for several observations regarding ethanol utilization and volatile fatty acid production in the rumen, its population size and growth characteristics suggest that it is not a significant contributor to ruminal metabolism in typical dairy cattle, although it may be a significant contributor to silage fermentation. The ability of unadapted cultures to produce substantial levels (12.8 g L(-1)) of caproic (hexanoic) acid in vitro suggests that this strain may have potential for industrial production of caproic acid.

Degradation of Cellulose and Hemicellulose by Ruminal Microorganisms.

As major structural components of plant cell walls, cellulose and hemicellulose are degraded and fermented by anaerobic microbes in the rumen to produce volatile fatty acids, the main nutrient source for the host. Cellulose degradation is carried out primarily by specialist bacteria, with additional contributions from protists and fungi, via a variety of mechanisms. Hemicelluloses are hydrolyzed by cellulolytic bacteria and by generalist, non-cellulolytic microbes, largely via extracellular enzymes. Cellulose hydrolysis follows first-order kinetics and its rate is limited by available substrate surface area. Nevertheless, its rate is at least an order of magnitude more rapid than in anaerobic digesters, due to near-obligatory adherence of microbial cells to the cellulose surface, and a lack of downstream inhibitory effects; in the host animal, fiber degradation rate is also enhanced by the unique process of rumination. Cellulolytic and hemicellulolytic microbes exhibit intense competition and amensalism, but they also display mutualistic interactions with microbes at other trophic levels. Collectively, the fiber-degrading community of the rumen displays functional redundancy, partial niche overlap, and convergence of catabolic pathways that all contribute to stability of the ruminal fermentation. The superior hydrolytic and fermentative capabilities of ruminal fiber degraders make them promising candidates for several fermentation technologies.

Microbial communities for valorizing biomass using the carboxylate platform to produce volatile fatty acids: A review.

The carboxylate platform employs a diverse microbial consortium of anaerobes in which the methanogens are inhibited. Nearly all biomass components are digested to a mixture of C1-C8 monocarboxylic acids and their corresponding salts. The methane-arrested anaerobic digestion proceeds readily without needing to sterilize biomass or equipment. It accepts a wide range of feedstocks (e.g., agricultural residues, municipal solid waste, sewage sludge, animal manure, food waste, algae, and energy crops), and produces high product yields. This review highlights several important aspects of the platform, including its thermodynamic underpinnings, influences of inoculum source and operating conditions on product formation, and downstream chemical processes that convert the carboxylates to hydrocarbon fuels and oxygenated chemicals. This review further establishes the carboxylate platform as a viable and economical route to industrial biomass utilization.

Complete genome of the cellulolytic ruminal bacterium Ruminococcus albus 7.

Ruminococcus albus 7 is a highly cellulolytic ruminal bacterium that is a member of the phylum Firmicutes. Here, we describe the complete genome of this microbe. This genome will be useful for rumen microbiology and cellulosome biology and in biofuel production, as one of its major fermentation products is ethanol.

Effects of preservation of rumen inoculum on volatile fatty acids production and the community dynamics during batch fermentation of fruit pomace.

Rumen fluid (RF) as inocula is useful for evaluating biomass digestibility and has potential for producing volatile fatty acids (VFA) via the carboxylate platform. However, RF is not readily available, necessitating evaluation of potential preservation methods. Glycerol (50% v/v) and DMSO (5% v/v) were used to preserve rumen inocula for 3 months at -80 °C. Effects of cryo-preservation on digestibility, VFA production and community composition with ß-diversity distance metrics were compared to fresh RF using apple, citrus and grape pomace as substrates. For all substrates, DMSO cryo-preserved rumen digestibility parameters, VFA yield and product distribution were more significantly comparable to fresh RF (P > 0.05) than was glycerol cryo-preserved RF. Similarly, ß-diversity coefficient (unweighted unifrac) between DMSO cryo-preserved RF and fresh RF was 0.250 while the coefficient was 0.359 for the glycerol cryo-preserved RF compared to fresh RF. This showed that a DMSO cryo-preserved RF is less affected by preservation effects and is a more promising alternative to fresh RF.

ARISA analysis of ruminal bacterial community dynamics in lactating dairy cows during the feeding cycle.

The bovine rumen undergoes substantial changes in environmental conditions during the animal's feeding cycle, but the effects of these changes on microbial populations have not been examined systematically. Two dairy cows fed a mixed forage/concentrate ration at 12 h intervals over 4 feeding cycles displayed substantial changes in ruminal pH and volatile fatty acid (VFA) concentrations. Automated ribosomal intergenic spacer analysis (ARISA) of solid- and liquid-associated bacterial populations in samples collected at 2, 4, 6, 9, and 12 h after feeding revealed a high degree of bacterial diversity. A total of 155 different amplicon lengths (ALs) were detected across all 83 samples, and 11-74 detected per sample. A substantial proportion (11%) of the ALs was detected in one cow but not in the other. The proportions of ALs that were detected only in the liquid phase or the solid phase were 13.5% and 1.9%, respectively. Correspondence analysis indicated that bacterial community composition differed between cows and between solid or liquid phases, but overall the solid-associated population displayed less change in composition within and across feeding cycles. The data support the notion that cows fed the same diets can have substantial differences in bacterial community composition, and that the solids-associated (biofilm) communities display greater stability than do associated planktonic communities.

Plant species composition and biofuel yields of conservation grasslands.

Marginal croplands, such as those in the Conservation Reserve Program (CRP), have been suggested as a source of biomass for biofuel production. However, little is known about the composition of plant species on these conservation grasslands or their potential for ethanol production. Our objective was to assess the potential of CRP and other conservation grasslands for biofuel production, describing the relationships of plant species richness and tall native C4 prairie grass abundance with plant chemical composition and the resulting potential ethanol yield. We determined plant species composition and diversity at multiple scales with the modified Whittaker plot technique, aboveground biomass, plant chemical composition, and potential ethanol yield at 34 sites across the major ecological regions of the northeastern USA. Conservation grasslands with higher numbers of plant species had lower biomass yields and a lower ethanol yield per unit biomass compared with sites with fewer species. Thus, biofuel yield per unit land area decreased by 77% as plant species richness increased from 3 to 12.8 species per m2. We found that, as tall native C4 prairie grass abundance increased from 1.7% to 81.6%, the number of plant species decreased and aboveground biomass per unit land area and ethanol yield per unit biomass increased resulting in a 500% increased biofuel yield per unit land area. Plant species richness and composition are key determinants of biomass and ethanol yields from conservation grasslands and have implications for low-input high-diversity systems. Designing systems to include a large proportion of species with undesirable fermentation characteristics could reduce ethanol yields.

Shifts in fermentation end products and bacterial community composition in long-term, sequentially transferred in vitro ruminal enrichment cultures fed switchgrass with and without ethanol as a co-substrate.

In vitro ruminal fermentations resemble in vivo fermentations with respect to substrate consumption and distribution of fermentation products in short term (1-5 d) incubations. However, little is known regarding changes in in vitro fermentations over prolonged incubation or multiple transfers. Gas production, pH, fermentation products, and bacterial community composition were examined in duplicate in vitro fermentations of switchgrass plus distillers grains that were transferred at 3-4 d intervals over 900 d. Additionally, duplicate fermentations inoculated from 160 d-old enrichments into the same medium but supplemented with ethanol, and transferred at 3-4 d over a 730 d period were characterized. SWG and SWG + E fermentation showed marked differences in community composition, pH, total product concentrations and ratios, relative to each other and to the original inoculum. The results have implications for the use of ruminal inocula for industrial production of short- and medium-chain fatty acids via the carboxylate platform.