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.     

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.     

Studies on the rumen flagellate Sphaeromonas communis.

The rumen flagellate Sphaeromonas communis showed a significant increase in population density 1 to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The life-history of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore by zoospores. Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 degrees C, presence of CO2, absnece of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absnece of bacteria and other flagellates.     

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.     

Studies on the rumen flagellate Neocallimastix frontalis.

The vast increase in the population density of the rumen flagellate Neocallimastix frontalis shortly after the host animal has commenced eating is caused by stimulation of a reproductive body on a vegetative phase of the organism to differentiate and liberate the flagellates. The stimulant is a component of the host's diet. The vegetative stage of N. frontalis bears a strong morphological resemblance to that of certain species of aquatic phycomycete fungi, and consists of a reproductive body borne on a single, much branched rhizoid. The flagellates liberated in vivo within 15 to 45 min of feeding lose their motility within I h and develop into the vegetative phase, thus producing a rapid decrease in population density of the flagellates. Conditions for maximum flagellate production are similar to those occurring in the rumen: pH 6-5, 39 degrees C, absence of O2, presence of CO2. Differentiation of the reproductive body is inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The organism was cultured in vitro in an undefined medium in the absence of bacteria or other flagellates.     

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.     

Alkaline phosphatase activity of rumen bacteria.

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Pectinolytic enzymes of large rumen treponemes.

Large spiral organisms isolated from the rumen of cattle produced and released into the external environment a complex of pectinolytic enzymes, consisting mainly of poly(1,4-alpha-D-galacturonide) lyase (EC 4.2.2.2, formerly EC 4.2.99.3), most active at pH 8.0 to 9.0, and another enzyme acting at pH below 7.0, probably a poly(1,4-alpha-D-galacturonide) glycanohydrolase (EC 3.2.1.15). The mixture of enzymes degraded polygalacturonate to saturated and unsaturated monogalacturonates as the end products. A pectin pectylhydrolase (pectinesterase) (EC 3.1.1.11) was also present in the clarified cultures.     

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.     

Pectinolytic enzymes of large rumen treponemes.

Large spiral organisms isolated from the rumen of cattle produced and released into the external environment a complex of pectinolytic enzymes, consisting mainly of poly(1,4-alpha-D-galacturonide) lyase (EC 4.2.2.2, formerly EC 4.2.99.3), most active at pH 8.0 to 9.0, and another enzyme acting at pH below 7.0, probably a poly(1,4-alpha-D-galacturonide) glycanohydrolase (EC 3.2.1.15). The mixture of enzymes degraded polygalacturonate to saturated and unsaturated monogalacturonates as the end products. A pectin pectylhydrolase (pectinesterase) (EC 3.1.1.11) was also present in the clarified cultures.