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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 β-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, β-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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 β-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.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mixed bacterial communities from the rumen ferment cellulosic biomass primarily to C2-C4 volatile fatty acids, and perform only limited chain extension to produce C5 (valeric) and C6 (caproic) acids. The aim of this study was to increase production of caproate and valerate in short-term in vitro incubations. Co-culture of mixed ruminal microbes with a rumen-derived strain of the bacterium Clostridium kluyveri converted cellulosic biomass (alfalfa stems or switchgrass herbage) plus ethanol to VFA mixtures that include valeric and caproic acids as the major fermentation products over a 48-72h run time. Concentrations of caproate reached 6.1gL(-1), similar to or greater than those reported in most conventional carboxylate fermentations that employ substantially longer run times., (Published by Elsevier Ltd.)

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.

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.

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.

"Extraruminal" fermentations employing in vitro incubation of mixed ruminal bacterial consortia, are capable of converting a complex array of biomass materials to mixtures of volatile fatty acids (VFA), methane, and carbon dioxide. Most of the potential energy in the biomass feedstock is retained in the VFA products, which are potential reactants for electrochemical conversion to hydrocarbon fuels. Quantitative data on VFA yields and proportions from biomass components are necessary for determining industrial feasibility, but such measurements have not been systematically reported. VFA yields and proportions were determined for a variety of carbohydrates, proteins and nucleic acids. Carbohydrates yielded primarily acetic and propionic acids, while proteins also yielded a more favorable product mix (longer average chain length and branched chain VFAs). Addition of certain co-substrates (e.g., glycerol) favorably improved the VFA product mix. The results have implications for hydrocarbon fuel generation from biomass materials by hybrid fermentation/chemical processes., (Published by Elsevier Ltd.)

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., (Published by Elsevier Ltd.)

Ruminant animals digest cellulose via a symbiotic relationship with ruminal microorganisms. Because feedstuffs only remain in the rumen for a short time, the rate of cellulose digestion must be very rapid. This speed is facilitated by rumination, a process that returns food to the mouth to be rechewed. By decreasing particle size, the cellulose surface area can be increased by up to 10(6)-fold. The amount of cellulose digested is then a function of two competing rates, namely the digestion rate (K(d)) and the rate of passage of solids from the rumen (K(p)). Estimation of bacterial growth on cellulose is complicated by several factors: (1) energy must be expended for maintenance and growth of the cells, (2) only adherent cells are capable of degrading cellulose and (3) adherent cells can provide nonadherent cells with cellodextrins. Additionally, when ruminants are fed large amounts of cereal grain along with fiber, ruminal pH can decrease to a point where cellulolytic bacteria no longer grow. A dynamic model based on STELLA software is presented. This model evaluates all of the major aspects of ruminal cellulose degradation: (1) ingestion, digestion and passage of feed particles, (2) maintenance and growth of cellulolytic bacteria and (3) pH effects.

Real-time polymerase chain reaction (PCR) was used to quantify 16 procaryotic taxa in the rumina of two lactating dairy cows following supply and subsequent withdrawal of the feed additive monensin (13.9 mg/kg of diet dry matter) in a high-starch, silage-based ration. PCR was conducted on DNA from rumen samples collected 6 h post feeding on two successive days before monensin supplementation, after 30 days of monensin supplementation, and at six weekly intervals after monensin withdrawal. Mean values of relative population size (RPS, the percent of bacterial 16S rRNA copy number) for genus Prevotella increased (P < 0.05) from 41.8% without monensin to 49.2% with monensin and declined to 42.5% after monensin withdrawal. Mean RPS values for two biohydrogenating species (Megasphaera elsdenii and Butyrivibrio fibrisolvens) were low (<0.4%) and declined several-fold in response to monensin. Mean RPS values for the biohydrogenating species Eubacterium ruminantium, four cellulolytic species, four starch- or dextrin-fermenting species, and Domain Archaea were not altered (P > 0.10) upon monensin feeding or withdrawal. The data suggest that monensin in high-starch diets does not suppress populations of classical ruminal Gram-positive bacteria or the availability of H2, though it may affect bacteria involved in biohydrogenation of lipids that regulate bovine mammary lipogenesis.

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.

Cattle generally avoid grazing leafy spurge (LS; Euphorbia esula), whereas sheep and goats will often eat it. Understanding metabolism of toxic phytochemicals in LS by bovine rumen microflora may help explain why cattle often develop aversions to LS after initially eating it. Toxicity of LS compounds after in vitro fermentation with normal vs. antibiotic-modified bovine rumen digesta was evaluated at different lengths of fermentation. Levels of toxic and aversion-inducing ingenols were determined for fermented and nonfermented mixtures of LS and bovine rumen digesta, and the toxicity of an aversion-inducing extract of LS to rumen microbial species that are common in cattle, sheep, and goats was evaluated. Fermentation of LS with bovine digesta increased the toxicity of extracted compounds. Introduction of neomycin (an antibiotic that preferentially inhibits gram-negative bacteria) into the LS and bovine rumen digesta mixtures did not appear to affect toxicities regardless of fermentation length. Levels of ingenol were observed in LS and bovine digesta mixtures (both fermented and nonfermented) that were consistent with levels of ingenols reported for LS. Finally, a toxic extract of LS had little or no negative effect on the growth of several common species of rumen bacteria. The results indicate that LS is not generally toxic to the ruminal bacteria, but that microbial activity in the rumen may be responsible for enhancing LS toxicity to cattle.

An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The ruminal microbial community is remarkably diverse, containing 100s of different bacterial and archaeal species, plus many species of fungi and protozoa. Molecular studies have identified a "core microbiome" dominated by phyla Firmicutes and Bacteroidetes, but also containing many other taxa. The rumen provides an ideal laboratory for studies on microbial ecology and the demonstration of ecological principles. In particular, the microbial community demonstrates both redundancy (overlap of function among multiple species) and resilience (resistance to, and capacity to recover from, perturbation). These twin properties provide remarkable stability that maintains digestive function for the host across a range of feeding and management conditions, but they also provide a challenge to engineering the rumen for improved function (e.g., improved fiber utilization or decreased methane production). Direct ruminal dosing or feeding of probiotic strains often fails to establish the added strains, due to intensive competition and amensalism from the indigenous residents that are well-adapted to the historical conditions within each rumen. Known exceptions include introduced strains that can fill otherwise unoccupied niches, as in the case of specialist bacteria that degrade phytotoxins such as mimosine or fluoroacetate. An additional complicating factor in manipulating the ruminal fermentation is the individuality or host specificity of the microbiota, in which individual animals contain a particular community whose species composition is capable of reconstituting itself, even following a neartotal exchange of ruminal contents from another herd mate maintained on the same diet. Elucidation of the interactions between the microbial community and the individual host that establish and maintain this specificity may provide insights into why individual hosts vary in production metrics (e.g., feed efficiency or milk fat synthesis), and how to improve herd performance. [ABSTRACT FROM AUTHOR]

Abstract: Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels. [Copyright &y& Elsevier]