Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum . increased the extent of microcrystalline cellulose degradation by 10·12% and 7·96%, respectively. Biomass yield in co-cultures was increased by 89·9% and 59·4% for M. elsdenii and E. limosum . Y_cellulose for fungus alone was 52·29 g dry matter mol^-1 glucose. These values were 64·93 and 55·92 g mol^-1 glucose unit in co-culture with M. elsdenii and E. limosum , respectively.     

Glucose and lactate catabolism by bacteria of the pig large intestine and sheep rumen as assessed by 13C nuclear magnetic resonance.

The fermentation of [1-13C] glucose and [3-13C]lactate by bacteria isolated from sheep rumen and pig large intestine was compared by the nuclear magnetic resonance (NMR) technique. Washed cell suspensions were incubated directly in the NMR spectrometer and spectra were recorded every 10 min after injection of the labelled substrates. The results showed large differences in the fermentation patterns between rumen and hindgut bacteria. The latter pattern indicated a greater ability for formation and fermentation of lactate than that of the rumen. Moreover, with both substrates the amount of propionate formed via the acrylate pathway was always greater with hindgut than with rumen bacteria, 50% and 20% of the total, respectively.     

Enumeration of Megasphaera elsdenii in rumen contents by real-time Taq nuclease assay

AIMS: To develop a real-time Taq nuclease assay (TNA) to enable the in vivo enumeration of Megasphaera elsdenii. METHODS AND RESULTS: Megasphaera elsdenii YE34 was phenotypically characteristic of the species and had 16S rDNA sequence similarity of 98% to previously described isolates. Calibration of the number of cells of M. elsdenii against the cycle threshold of fluorescent dye release gave a straight-line relationship with a correlation coefficient approximating unity. The specificity of the assay for M. elsdenii was confirmed by performing it against a panel of 24 heterogeneous, mainly ruminal bacteria. Megasphaera elsdenii was not detected in ruminal contents from a pasture-fed steer but was readily detected 2 and 50 h after the probiotic introduction of the bacterium into the rumen. CONCLUSIONS: Real-time TNA has provided a sensitive and specific means of enumerating the M. elsdenii population in rumen contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Megasphaera elsdenii is an important lactate-degrading ruminal bacterium that has been selected for probiotic use to prevent acidosis and enhance starch utilization in grain-fed cattle. The assay developed in this study provides a tool for determining the ability of probiotically-introduced M. elsdenii to establish useful populations in the rumen.     

In vitro ruminal fermentation of organic acids common in forage.

Mixed rumen bacteria from cows fed either timothy hay or a 60% concentrate were incubated with 7.5 mM citrate, trans-aconitate, malate, malonate, quinate, and shikimate. Citrate, trans-aconitate, and malate were fermented at faster rates than malonate, quinate, and shikimate. Acetate was the primary fermentation product for all six acids. Quinate and shikimate fermentations gave rist to butyrate, whereas malate and malonate produced significant amounts of propionic acid. High-pressure liquid chromatography of fermentation products from trans-aconitate incubations revealed a compound that was subsequently identified as tricarballylate. As much as 40% of the trans-aconitate acid was converted to tricarballylate, and tricarballylate was fermented slowly. The slow rate of tricarballylate metabolism by mixed rumen bacteria and its potential as a magnesium chelator suggest that tricarballylate formation could be an important factor in the hypomagnesemia that leads to grass tetany.     

Fermentative degradation of glutarate via decarboxylation by newly isolated strictly anaerobic bacteria

Two strains of new strictly anaerobic, gramnegative bacteria were enriched and isolated from a freshwater (strain WoG13) and a saltwater (strain CuG11) anoxic sediment with glutarate as sole energy source. Strain WoG13 formed spores whereas strain CuG11 did not. Both strains were rod-shaped, motile bacteria growing in carbonate-buffered, sulfide-reduced mineral medium supplemented with 2% of rumen fluid. Both strains fermented glutarate to butyrate, isobutyrate, CO₂, and small amounts of acetate. With methylsuccinate, the same products were formed, and succinate was fermented to propionate and CO₂. No sugars, amino acids or other organic acids were used as substrates. Molar growth yields (Ys) were very small (0.5–0.9 g cell dry mass/mol dicarboxylate). Cells of strain WoG13 contained no cytochromes, and the DNA base ratio was 49.0±1.4 mol% guanine-plus-cytosine. Enzyme activities involved in glutarate degradation could bedemonstrated in cell-free extracts of strain WoG13. A pathway of glutarate fermentation via decarboxylation of glutaconyl-CoA to crotonyl-CoA is suggested which forms butyrate and partly isobutyrate by subsequent isomerization.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

Detection of acrylic acid formation in Megasphaera elsdenii in the presence of 3-butynoic acid

Summary The formation of acrylic acid from lactic acid in the anaerobic rumen bacterium Megasphaera elsdenii was detected in the presence of 3-butynoic acid. While the major end products of lactic acid fermentation in the absence of the inhibitor were propionate, acetate, valerate, and butyrate, the presence of 3-butynoic acid led to the production of propionate, acetate, acrylate, and butyrate. An improvement in the chemical synthesis and purification of 3-butynoic acid was developed.     

Two Pathways of Glutamate Fermentation by Anaerobic Bacteria

Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia-the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-(14)C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.     

Importance of the Isovalerate Carboxylation Pathway of Leucine Biosynthesis in the Rumen

Certain anaerobic ruminal bacteria synthesize the leucine carbon skeleton by use of a pathway different from that described in other microorganisms. These organisms carboxylate the intact carbon skeleton of isovalerate, synthesizing leucine-2-C(14) from isovalerate-1-C(14). Strains of Bacteroides ruminicola and Peptostreptococcus elsdenii were like Ruminococcus flavefaciens in that they incorporated appreciable amounts of C(14) from isovalerate-1-C(14) into cellular protein and in that the only labeled amino acid found was leucine. The specific activity of beta-isopropylmalate dehydrogenase in extracts from R. flavefaciens and from the mixed bacterial population from the rumen was very low as compared with the specific activity of this enzyme in extracts from Escherichia coli. This suggests that the pathway of leucine biosynthesis that operates in many aerobic and facultative microorganisms is not the major pathway in rumen bacteria. This was supported by the finding that after fermentation of whole rumen contents with acetate-2-C(14), leucine from the bacterial cells had a specific activity lower than one would expect if acetate was incorporated directly into carbons 1 and 2 of leucine.     

Transport of butyrate across the isolated bovine rumen epithelium--interaction with sodium, chloride and bicarbonate

The Ussing chamber technique was used for studying unidirectional fluxes of 14C-butyrate across the bovine rumen epithelium in vitro. Significant amounts of butyrate were absorbed across the bovine rumen epithelium in vitro, without any external driving force. The paracellular pathway was quantitatively insignificant. The transcellular pathway was predominately voltage-insensitive. The serosal to mucosal (SM) pathway was regulated by mass action, whereas the mucosal to serosal (MS) pathway further includes a saturable process, which accounted for 30 to 55% of the MS flux. The studied transport process for 14C-butyrate across the epithelium could include metabolic processes and transport of 14C-labelled butyrate metabolites. The transport of butyrate interacted with Na+, Cl- and HCO3-, and there was a linear relationship between butyrate and sodium net transport. Lowering the sodium concentration from 140 to 10 mmol l-1 decreased the butyrate MS flux significantly. Amiloride (1 mmol l-1) did, however, not reduce the butyrate flux significantly. Chloride concentration in itself did not seem to influence the transport of butyrate, but chloride-free conditions tended to increase the MS and SM flux of butyrate by a DIDS-sensitive pathway. DIDS (bilateral 0.5 mmol l-1) did further decrease the butyrate SM flux significantly at all chloride concentrations. Removing bicarbonate from the experimental solutions decreased the MS and increased the SM flux of butyrate significantly, and abolished net butyrate flux. There were no significant effects of the carbonic anhydrase inhibitor Acetazolamide (bilateral 1.0 mmol l-1). The results can be explained by a model where butyrate and butyrate metabolites are transported both by passive diffusion and by an electroneutral anion-exchange with bicarbonate. The model couples sodium and butyrate via CO2 from metabolism of butyrate, and intracellular pH.     

Interactions between rumen amylolytic and lactate-utilizing bacteria in growth on starch

The growth and metabolism of the rumen amylolytic bacteria Streptococcus bovis, Butyrivibrio fibrisolvens and Bacteroides ruminicola, growing in pure cultures and co-cultures with the rumen lactilytic bacteria Megasphaera elsdenii and Veillonella alcalescens were followed. The interaction of amylolytic bacteria with V. alcalescens represents a simple food chain. The interaction with M. elsdenii is more complex, since there is a simultaneous competition for products of the starch degradation.     

Interactions between rumen amylolytic and lactate-utilizing bacteria in growth on starch

The growth and metabolism of the rumen amylolytic bacteria Streptococcus bovis, Butyrivibrio fibrisolvens and Bacteroides ruminicola , growing in pure cultures and co-cultures with the rumen lactilytic bacteria Megasphera elsdenii and Veillonella alcalescens were followed. The interaction of amylolytic bacteria with V. alcalescens represents a simple food chain. The interaction with M. elsdenii is more complex, since there is a simultaneous competition for products of the starch degradation.     

Lactate and Acrylate Metabolism by Megasphaera elsdenii under Batch and Steady-State Conditions

The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied under batch and steady-state conditions. Under batch conditions, lactate was converted to acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl propenoate and 3-butenoate containing a terminal double bond were similarly converted into their respective saturated acids (isobutyrate and butyrate), while crotonate and lactate analogues 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. As the acrylate concentration in the feed was increased, butyrate and hydrogen formation decreased and propionate was increasingly generated, while the calculated ATP yield was unchanged. M. elsdenii metabolism differs substantially under batch and steady-state conditions. The results support the conclusion that propionate is not formed during lactate-limited steady-state growth because of the absence of this substrate to drive the formation of lactyl coenzyme A (CoA) via propionyl-CoA transferase. Acrylate and acrylate analogues are reduced under both batch and steady-state growth conditions after first being converted to thioesters via propionyl-CoA transferase. Our findings demonstrate the central role that CoA transferase activity plays in the utilization of acids by M. elsdenii and allows us to propose a modified acrylate pathway for M. elsdenii.     

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.     

On the contribution of the acrylate pathway to the formation of propionate from lactate in the rumen of cattle

The effect of chloral hydrate, an inhibitor of methanogenesis, on the participation of the acrylate pathway in the formation of propionate from lactate in rumen contents of cattle was studied in vitro. Addition of chloral hydrate resulted in only a small stimulation of the acrylate pathway, much lower than the stimulation of propionate production by chloral hydrate. This means that the flux of carbon through both the acrylate and the dicarboxylic acid pathway is increased during chloral hydrate feeding.The influence of time of sampling after feeding on the contribution of the acrylate pathway was studied in a separate experiment. A marked drop in the participation of the acrylate pathway in propionate formation from lactate during at least 2 h after feeding was observed, whereafter a rapid rise to prefeeding levels occurred.     

The effect of rumen epithelial development on metabolic activities and ketogenesis by the tissue in vitro.

1. An investigation was made on oxygen consumption, glucose and lactate uptake and ketogenesis from butyrate by rumen epithelium in vitro from lambs at various stages of development. 2. Oxygen uptake was decreased by about 35% and glucose uptake by about 90% between 2 weeks and 1/2 year of age. 3. The uptake of L-lactate and the utilization of butyrate as a substrate for respiration were increased during epithelial development. 4. The production of D(-)-3-hydroxybutyrate and acetoacetate from butyrate by the epithelium was largely increased between 4 to 10 weeks of age, independently of rumen fermentation. 5. A synergistic effect of glucose on the production of D(-)-3-hydroxybutyrate and on total ketone bodies from butyrate by the epithelium was observed. It accounted to 40-80% over butyrate depending on the stage of epithelial development.     

Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets

Abstract Samples of rumen ingesta from two rumen-fistulated dairy cows fed grass silage-based diets were examined for numbers and types of bacteria that developed colonies on rumen fluid-agar media designated to support the growth of (a) a wide range of species, (b) cellulolytic bacteria, (c) lactate-fermenting bacteria, (d) non-fermentative bacteria. The most numerous species was Bacteroides ruminicola followed by Butyrivibrio fibrisolvens . The most abundant cellulolytic species were Eubacterium cellulosolvens and Ruminococcus flavefaciens. Megasphaera elsdenii and Selenomonas ruminantium were important lactate fermenters but an unidentified bacterium that grew poorly on maintenance medium was by far the most numerous among bacteria isolated from lactate-containing medium. One strain remained sufficiently viable to show that it fermented lactate to propionate and acetate.     

Improvement of butanol fermentation by supplementation of butyric acid produced from a brown alga

This study investigated butanol fermentation using glucose and culture broth containing butyrate from the butyrate fermentation of a brown alga, Laminaria japonica. Prior to the use of the biologically-produced butyrate, the initial glucose in tryptone-yeast extract acetate (TYA) medium was first optimized for butanol fermentation using Clostridium saccharoperbutylacetonicum N1-4 ATCC 27021^T. Then, a commercially-acquired (synthetic) butyrate was supplemented to the TYA medium containing the optimal glucose concentration (around 30 and 60 g/L). According to the experimental results, the highest butanol carbon yield (0.580 C-mol/C-mol) was obtained from the fermentation of 36.65 g/L glucose and 7.29 g/L synthetic butyrate. Fermentation of a similar amount of glucose (32.28 g/L) in the absence of butyrate gave a butanol carbon yield of 0.402 C-mol/C-mol. For the experiment with fermented butyrate, a 100 g/L biomass of brown alga was fermented by Clostridium tyrobutyricum ATCC 25755 and the culture broth containing butyrate was used to prepare TYA medium after removing the bacterial cells. Fermentation using the synthetic butyrate and the biologically-produced butyrate (4.95 g/L) gave a comparable butanol concentration (13.23 g/L) and butanol carbon yield (0.513 C-mol/C-mol). Overall, this study proved that the addition of fermented butyrate from brown alga fermentation could be an effective way to improve butanol production. Furthermore, the reuse of spent medium and the absence of rigorous purification of the broth containing butyrate would lower the production cost of the fermentation.     

Methane production and diurnal variation measured in dairy cows and predicted from fermentation pattern and nutrient or carbon flow

Many feeding trials have been conducted to quantify enteric methane (CH₄) production in ruminants. Although a relationship between diet composition, rumen fermentation and CH₄ production is generally accepted, the efforts to quantify this relationship within the same experiment remain scarce. In the present study, a data set was compiled from the results of three intensive respiration chamber trials with lactating rumen and intestinal fistulated Holstein cows, including measurements of rumen and intestinal digestion, rumen fermentation parameters and CH₄ production. Two approaches were used to calculate CH₄ from observations: (1) a rumen organic matter (OM) balance was derived from OM intake and duodenal organic matter flow (DOM) distinguishing various nutrients and (2) a rumen carbon balance was derived from carbon intake and duodenal carbon flow (DCARB). Duodenal flow was corrected for endogenous matter, and contribution of fermentation in the large intestine was accounted for. Hydrogen (H₂) arising from fermentation was calculated using the fermentation pattern measured in rumen fluid. CH₄ was calculated from H₂ production corrected for H₂ use with biohydrogenation of fatty acids. The DOM model overestimated CH₄/kg dry matter intake (DMI) by 6.1% (R²=0.36) and the DCARB model underestimated CH₄/kg DMI by 0.4% (R²=0.43). A stepwise regression of the difference between measured and calculated daily CH₄ production was conducted to examine explanations for the deviance. Dietary carbohydrate composition and rumen carbohydrate digestion were the main sources of inaccuracies for both models. Furthermore, differences were related to rumen ammonia concentration with the DOM model and to rumen pH and dietary fat with the DCARB model. Adding these parameters to the models and performing a multiple regression against observed daily CH₄ production resulted in R² of 0.66 and 0.72 for DOM and DCARB models, respectively. The diurnal pattern of CH₄ production followed that of rumen volatile fatty acid (VFA) concentration and the CH₄ to CO₂ production ratio, but was inverse to rumen pH and the rumen hydrogen balance calculated from 4×(acetate+butyrate)/2×(propionate+valerate). In conclusion, the amount of feed fermented was the most important factor determining variations in CH₄ production between animals, diets and during the day. Interactions between feed components, VFA absorption rates and variation between animals seemed to be factors that were complicating the accurate prediction of CH₄. Using a ruminal carbon balance appeared to predict CH₄ production just as well as calculations based on rumen digestion of individual nutrients.     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.