Desulfovibrio of the sheep rumen.

A sulfate-reducing bacterium has been isolated in pure culture from sheep rumen contents. Its properties agree in all respects tested with those ascribed to Desulfovibrio desulfuricans. The populations observed (about 10(8)/ml) are sufficient to account for published rates of ruminal sulfide production.     

Fermentation of methanol in the sheep rumen.

Sheep fed a hay-concentrate diet were adapted to pectin administration and ruminal infusion of methanol. Both treatments resulted in a strong increase in the rate of methanogenesis from methanol. Quantitative data show that methanol was exclusively converted into methane. Treatments did not influence ruminal volatile fatty acid percentages.     

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.     

Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen.

The rumen flagellat Piromonas communis is the zoospore of a phycomycete fungus inhabiting the rumen. Zoosporogenesis was stimulated by a dietary component (the inducer), and inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The zoospores showed taxis towards the tissues surrounding the inflorescence of Lolium perenne L. in the rumen, invading principally the stomata and damaged tissues. The zoospores germinated on this substratum and the rhizoids of the developing vegetative stage penetrated the tissue, taking up C14 from labelled plant material, which was incorporated into the fungal cells. The conditions for maximum flagellate production (39 degrees C, pH 6-0 to 7-0, high concentration of CO2, absence of O2) resembled those found in the rumen. The organism was cultured in an undefined medium in vitro in the absence of other flagellates.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

Presence of soluble lignin-carbohydrate complexes in the bovine rumen.

The cell-free rumen liquor of a steer on a diet of spear grass has been shown to contain macromolecular substances in which carbohydrates and lignin-derived compounds are covalently bound to each other. The lignin-carbohydrate complexes are soluble at pH 7 or higher, but precipitate at pH 3. At the latter pH, small amounts of a polymer, assumed to be glycoprotein, remain in solution. Some of the lignin-carbohydrate linkages are broken by treatment with alkali. Treatment with 50mM sulphuric acid for a few minutes at room temperature converts part of the complex into an acetone-soluble product, which still contains both carbohydrate and lignin-derived compounds. The formation of soluble lignin-carbohydrate complexes by the action of rumen micro-organisms on the grass may account for the dissolution (and hence the apparent digestion) of about half of the total lignin-intake.     

Thiomolybdates in rumen contents and rumen cultures

Examination of direct and (Cu)-difference spectra of i) the aqueous supernatants of in vitro cultures of bovine rumen contents incubated with MoO42- and potential sources of S2- and ii) samples drawn directly from the rumen of animals receiving high Mo diets yielded evidence of the presence of thiomolybdates. Only MoS42- was detected in the soluble phase of in vitro cultures. Although intense and variable background absorbance precluded full characterization of thiomolybdate species in samples drawn directly from the rumen, both spectral data and the biochemical and clinical responses of animals given high Mo diets were consistent with the conclusion that MoS42- rather than MoOS32- was the predominant thiomolybdate species present in the aqueous phase. Addition of Ca2+ either to rumen cultures before incubation or as a supplement to diets high in MoO42- content inhibited the appearance of MoS42- in the aqueous phase. Evidence of the sequestration of MoS42- and MoOS32- by particulate or microbial fractions of rumen contents is considered in relation to the inhibitory action of Mo upon Cu absorption by ruminants.     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Physiological diversity of rumen spirochetes.

Bovine rumen fluid contained relatively large numbers of spirochetes capable of fermenting polymers commonly present in plant materials. Polymers such as xylan, pectin, and arabinogalactan served as fermentable substrates for the spirochetes, whereas cellulose did not. Furthermore, spirochetes cultured from rumen fluid utilized as growth substrates hydrolysis products of plant polymers (e.g., D-xylose, L-arabinose, D-galacturonic acid, D-glucuronic acid, cellobiose), but did not ferment amino acids. Viable cell counts of spirochetes capable of fermenting individual plant polymers or their hydrolysis products yielded minimum values ranging from 0.2 X 10(6) to 4 X 10(6) cells per ml of rumen fluid. Thirteen strains of rumen spirochetes were characterized in terms of their fermentation products from glucose, the guanine plus cytosine content of their DNA, their ultrastructure, and their ability to ferment pectin, starch, or arabinogalactan. Of the 13 strains, 6 fermented glucose mainly to formate, acetate, and succinate, whereas the remaining 7 strains did not produce succinate, but instead formed ethanol, in addition to formate and acetate. The succinate-forming strains had two periplasmic (axial) fibrils per cell, measured 0.2 to 0.3 by 5 to 8 micrograms, had a guanine plus cytosine content of the DNA ranging from 36 to 38 mol%, and lacked the ability to ferment pectin, starch, or arabinogalactan. The ethanol-forming strains had from 8 to more than 32 periplasmic fibrils per cell, tended to be larger in cell size than the succinate-forming strains, and had a guanine plus cytosine content of the DNA ranging from 41 to 54 mol%. Some of the ethanol-forming strains fermented pectin, starch, or arabinogalactan. The results of this study indicate that the bovine rumen is inhabited by a physiologically and morphologically diverse population of spirochetes. It is likely that these spirochetes contribute significantly to the degradation of plant materials ingested by the ruminants.     

Production of benzodiazepine-like compounds in bovine rumen.

The presence of benzodiazepine-like molecules was detected radioimmunologically in bovine rumen contents and in incubates of ruminal contents with homogenates of several common grasses. A similar production was found "in vivo" in samples obtained from a grazing cow with a rumen cannula.     

The pathway of ketogenesis in rumen epithelium of the sheep.

A method for the fractionation of sheep rumen epithelium with limited mitochondrial breakage is described. The distributions of the enzymes of the 3-hydroxy-3-methylglutaryl-CoA pathway of ketogenesis indicate that this process is exclusively mitochondrial. Enzyme activities are sufficient to account for the ketogenic rates found in vivo. The failure of (-)-hydroxycitrate to block ketogenic flux supports this view. 3-Hydroxybutyrate dehydrogenase activity is largely associated with particulate material in the mitochondrial fraction. ATP citrate lyase activity was found, with appreciable acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase in the cytoplasmic fraction.