Capacity of Polysaccharide Accumulation by Whole Rumen Bacteria

An optical density of a whole bacterial suspension, prepared from sheep rumen contents, increased very rapidly when the cells were incubated in a glucose-containing medium. This is largely due to the accumulation of intracellular polysaccharide(s) and appears to proceed without cell multiplication. The increase has an apparent relationship with feeding conditions of animals and reflects the availability of easily fermentable sugars for bacteria in the rumen. The data suggest that the ruminal fermentation proceeds under extremely low level of easily fermentable sugars.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

In Vitro Metabolism of the Stereoisomers of 2,6-Diaminopimelic Acid by Mixed Rumen Protozoa and Bacteria

Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria. Received: 16 March 1996 / Accepted: 14 May 1996     

Comparison of Substrate Affinities Among Several Rumen Bacteria: a Possible Determinant of Rumen Bacterial Competition

Five rumen bacteria, Selenomonas ruminantium, Bacteroides ruminicola, Megasphaera elsdenii, Streptococcus bovis, and Butyrivibrio fibrisolvens were grown in continuous culture. Estimates of substrate affinities were derived from Lineweaver-Burk plots of dilution rate versus substrate concentration. Each bacterium was grown on at least four of the six substrates: glucose, maltose, sucrose, cellobiose, xylose, and lactate. Wide variations in substrate affinities were seen among the substrates utilized by a species and among species for the same substrate. These wide differences indicate that substrate affinity may be a significant determinant of bacterial competition in the rumen where soluble substrate concentrations are often low. Growth of these bacteria in continuous culture did not always follow typical Michaelis-Menten kinetics. Inflated theoretical maximum growth rates and non-linear Lineweaver-Burk plots were sometimes seen. Maintenance energy expenditures and limitation of growth rate by factors other than substrate concentration (i.e., protein synthesis) are discussed as possible determinants of these deviations.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

In Vitro Catabolism of Histidine by Mixed Rumen Bacteriaand Protozoa

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39°C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 μmol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated. Received: 23 May 2000 / Accepted: 31 July 2000     

Conversion of Lysine to Pipecolic Acid by Rumen Ciliate Protozoa

Addition of lysine to the culture medium of rumen ciliates increased the amount of pipecolate in the medium to the level over the control.

l-Lysine- U-¹⁴C was partly incorporated into ciliate protein unchanged, and partly converted to radioactive pipecolate. The autoradiogram of amino acids in supernatant fluid of the medium revealed the traces of two unidentified spots other than lysine and pipecolate as a main metabolite.

Since the radioactive pipecolate was not changed further more by ciliates, it seemed to be the end product in lysine degradation.

After addition of l-lysine-α,ε-¹⁵N to the medium, ¹⁵N was detected mainly in the fractions of pipecolate and ammonia and a little in that of glutamate, alanine and aspartate of the medium.     

Hemicellulose-degrading Enzymes Synthesized by Rumen Bacteria

Over 100 bacterial cultures isolated from ovine rumen contents by enrichment techniques in polysaccharide-containing media were examined for the ability to degrade plant cell wall structural polysaccharides. Approximately two-thirds of the isolates retained were Gram negative and amongst the coccoid isolates diplococci predominated. Many of the rods examined were piliated and/or flagellated. Thirty of the more active xylan- and arabinan-degrading isolates were examined for both the production and activity of the principal polysaccharidase and glycosidase enzymes associated with plant cell wall polysaccharide hydrolysis, following culture in a hemicellulose containing growth medium. The wide range of glycosidase activities detected in these hemicellulolytic isolates was evidence for their rôle in the breakdown of dietary polysaccharides in the rumen.     

Cultivation of Rumen Protozoa. I. Factors Influencing the Cultivation of Rumen Oligotrich Protozoa "In Vitro"

SYNOPSIS. Factors influencing the cultivation of entodinia in vitro have been studied. It was found that removal of particulate material, culture division, or a combination of both resulted in similar maximum protozoal concentrations. At the same time, untreated culture concentrations declined rapidly after reaching a maximum concentration at about the tenth day.
At concentrations of streptomycin greater than 25 μg per ml of media, increasing the level of streptomycin extended the time required for a culture to attain a maximum protozoal concentration. A significant relationship (P>.01) was demonstrated between the starch concentration and the protozoal concentration, and it was found that various combinations of starch and streptomycin produced different relative protozoal concentrations in initial and established cultures. Implications arising from these results are discussed.     

Specificity of Rumen Ciliate Protozoa in Cattle and Sheep*

SYNOPSIS. Six protozoa-free sheep, 3 fed alfalfa hay and 3 fed a concentrate diet, were inoculated with rumen contents from a steer fed the same alfalfa hay. All 24 species of protozoa in the inoculum became established in the sheep fed alfalfa hay, while only 9 species established in the sheep fed concentrate. Percentage species composition in the alfalfa-fed sheep was fairly similar to that of the inoculum. Rumen volumes of the alfalfa hay-fed sheep were significantly higher than those of the concentrate-fed sheep; however, fluid turnover rates were similar. Total protozoan numbers per ml of rumen contents were significantly higher in the concentrate-fed sheep, but after adjustment for rumen volume, there was no significant difference in the total number of protozoa in the rumen.     

High Molecular Weight Protease in Rumen Ciliate Protozoa

Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein.     

Catabolism of Lysine by Mixed Rumen Bacteria

Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ ml of lysine was decomposed to give ether-soluble substances and CO₂ by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. δ-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did.     

Medium Without Rumen Fluid for Nonselective Enumeration and Isolation of Rumen Bacteria

Colony counts which approximated those in a habitat-simulating, rumen fluid-agar medium (RFM) were obtained in medium 10, a medium identical to the RFM except for the replacement of rumen fluid with 1.5 x 10(-6)m hemin, 0.2% Trypticase, 0.05% yeast extract, and a 6.6 x 10(-2)m volatile fatty acid mixture qualitatively and quantitatively similar to that in rumen fluid. Single deletion of Trypticase, yeast extract, or the volatile fatty acid mixture from medium 10 significantly reduced colony counts. Colony counts were also reduced when medium 10 was modified to contain higher concentrations of Trypticase or volatile fatty acids. Significant differences were found between colony counts obtained from diluted rumen contents of animals fed a cracked corn-urea diet, and the colony counts obtained from animals fed either a cracked corn-soyean oil meal or an alfalfa hay-grain diet. Qualitative differences were found between the predominant bacterial strains isolated from rumen contents of animals fed cracked corn diets and strains isolated from animals fed alfalfa hay-grain. Regardless of differences in the predominant flora associated with diet, medium 10 and the RFM supported growth of similar bacterial populations. The results show that medium 10 is suitable for enumeration and isolation of many predominant rumen bacteria.     

Competition between Food Particles and Rumen Bacteria in the Uptake of Long-chain Fatty Acids and Triglycerides

S ummary . Suspensions of mixed rumen bacteria were incubated with long-chain fatty acids and their corresponding triglycerides in the presence and absence of the food particles present in the rumen. The uptake of free fatty acids and triglycerides by the bacteria was due largely to physical adsorption. With increasing unsaturation of the free fatty acids, less adsorption occurred and in all instances the presence of food particles reduced greatly the extent to which the fatty acids were adsorbed on the bacteria. The triglycerides were adsorbed to the bacteria to a much less extent than their corresponding free fatty acids, more of the substrate remaining in the supernatant fraction. The presence of food particles had little effect on the extent to which the triglycerides became adsorbed to the bacteria, but reduced greatly the amount of triglyceride present in the supernatant fraction. The decrease in amount of free fatty acid associated with the bacteria and the decrease in the amount of triglyceride in the supernatant liquid when food particles were present, was wholly accounted for by the increase in amount of these substrates associated with the food particles. The implications of these findings with respect to the metabolism of lipids in the rumen is discussed.     

Lysis of Viable Rumen Bacteria in Bovine Rumen Fluid

Streptococcus bovis and Butyrivibrio sp. were labeled with thymidine-methyl-(3)H, washed, and resuspended in rumen fluid or rumen fluid fractions obtained from Holstein and Jersey cows fed alfalfa hay once daily. Factors affecting the lytic activity found in untreated rumen fluid were examined. Day to day variation and differences before and after feeding were observed for the same cow. There were also differences between cows on the same day. For a given rumen fluid, the rate of release of label was roughly proportional to the number of labeled cells present over a 100-fold range in concentration. Removal of protozoa largely abolished the lytic action of fresh rumen fluid for S. bovis, but some soluble lytic activity remained. Mixed rumen protozoa added to media containing labeled S. bovis caused label to appear in solution. In a sample of rumen fluid containing 4.3 x 10(4) protozoa/ml 5.2% of the S. bovis population were destroyed by protozoa per hr. The mean rate of destruction for 12 runs on whole rumen fluid was 8.7% per hr with a standard deviation of 6.05. Parallel experiments with Butyrivibrio indicated that soluble lytic factors were more important for this organism. They could be destroyed by autoclaving and were generated when viable rumen bacteria were resuspended in autoclaved rumen fluid. The lysis of S. bovis and Butyrivibrio, at equal cell densities, by mixed rumen protozoa was compared in 30% rumen fluid media, and Butyrivibrio appeared to be more readily lysed than S. bovis.     

瘤胃纤维素降解细菌的研究进展

反刍动物瘤胃是自然界中最有效的纤维素降解系统，其纤维素降解能力主要源于寄居于其中的纤维素降解细菌、真菌和原虫。其中，瘤胃纤维素降解细菌因数量庞大、种类繁多以及代谢途径丰富，在木质纤维素降解及利用方面发挥着重要作用。本文综述了国内外瘤胃纤维素降解细菌的种类，分析了瘤胃纤维素降解细菌的特性；阐述了瘤胃纤维素降解细菌通过纤维小体对纤维素的降解过程，以及瘤胃微生物之间的相互作用和相互制约关系；简述宏组学技术在开发新纤维素降解菌和新纤维素酶方面的应用，旨在为进一步研究纤维素降解细菌的降解机理，开发新的纤维素菌种和酶资源提供新的思路。     

In Vitro Production of Lysine from 2,2'-Diaminopimelic Acid by Rumen Protozoa

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu [12] and Masson & Ling [9] show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10⁵ protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.