The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen.

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 micrograms/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 micrograms/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2-7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 micrograms/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum, but not Entodinium spp. or Dip. affine, synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 micrograms/mg protein/min. If these reactions occur in the rumen in vivo, up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa.

Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum, ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus megaterium. Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bovis, although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro, although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa. Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.

The degradation of polygalacturonic acid by rumen ciliate protozoa.

The depolymerase activity of cell-free extracts of nine species of rumen ciliate protozoa and two mixed protozoal preparations, grown in vivo and in vitro, towards polygalacturonic acid was examined. The highest activity was found with Eremoplastron bovis and Ostracodinium obtusum bilobum while there was none in the spined or spineless forms of Entodinium caudatum and little in Polyplastron multivesticulatum. On the basis of the rapid drop in viscosity, inhibition by EDTA and the production of u.v.-absorbing material, the enzymes from all active species were designated as endopectate lyases (EC4.2.2.2) although some polygalacturonase may be present. Neither pectin nor polygalacturonic acid supported the survival or growth of any of the protozoal species tested.

The uptake and metabolism of bacteria, amino acids, glucose and starch by the spined and spineless forms of the rumen ciliate Entodinium caudatum.

Spined and spineless forms of Entodinium caudatum were obtained by growth in vivo in the presence and absence, respectively, of Entodinium bursa. Washed suspensions of both forms engulfed all the bacteria tested although the spined form took them up 1.3 to 1.9 times more rapidly per unit volume of protozoon than did the spineless form. Buytrivibrio fibrisolvens and Selenomonas ruminantium were rapidly digested by the spined form after engulfment. Free amino acids were taken up on average 3.1 times and glucose approximately 60 times faster per unit volume of protozoon by the spined form. Limited amounts of protein were synthesized by the spined form from glucose and starch but engulfed bacteria and, to a lesser extent, free amino acids were probably the prinicpal sources of protein for growth of both forms.

The cultivation of the rumen ciliate Entodinium bursa in the presence of Entodinium caudatum.

The rumen ciliate protozoon Entodinium bursa has been grown in vitro in the presence of bacteria and Entodinium caudatum for over a year at population densities of 100 to 200 ml-1. The medium contained potassium phosphate, prepared fresh rumen fluid, cysteine, wholemeal flour (or rice starch), dried grass and a culture of the spineless form of Entodinium caudatum. Entodinium bursa has an obligate requirement for this protozoon and died within 48 h in its absence. During growth from a 2% inoculum, the mean generation time of E. bursa was 6 h. Entodinium bursa engulfed 1-5 to 2-5 E. caudatum organisms h-1, and when E. caudatum was in excess it developed caudal spines for the first time in 17 years; these spined forms were engulfed much less readily than the spineless organisms.

The metabolism of starch, glucose, amino acids, purines, pyrimidines and bacteria by the rumen ciliate Polyplastron multivesiculatum.

The large rumen ciliate protozoon Polyplastron multivesiculatum grown in vitro engulfed a wide range of bacteria (from a population density of 10(9) bacteria ml(-1)) at a rate of 1500 to 137000 bacteria h(-1) protozoon(-1). No evidence was found for the preferential engulfment of bacteria of rumen origin. Except for Proteus mirabilis none of the bacteria were digested with the liberation of soluble materials into the medium. Glucose and amino acids were taken up rapidly by P. multivesiculatum compared with the rate of uptake by Entodinium caudatam. Glucose was incorporated into protozoal polysaccharide and into bacteria associated with the protozoa and was used for the synthesis of a wide range of amino acids. Evidence showed that bacteria and free amino acids at the concentrations found in the rumen could supply the protein requirements of the protozoa for division at least once each day.

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.

The cultivation of cellulolytic protozoa isolated from the rumen.

Conditions are described for the isolation from the rumen and subsequent growth of six species of cellulolytic protozoa: Enoploplastron triloricatum, Eudiplodinium maggii, Diploplastron affine, Epidinium ecaudatum caudatum, Diploidinium monacanthum and Diploidinium pentacanthum. The protozoa were grown in an atmosphere of 95% nitrogen plus 5% carbon dioxide, or pure carbon dioxide, in a potassium phosphate-rich medium containing cysteine, sometimes 10% prepared fresh rumen fluid, and daily additions of powdered dried grass. Population densities of 10 to 6000 protozoa/ml (depending on the species) were obtained in cultures that were diluted with fresh medium twice each week. Extracts of these protozoa digested a [14C]cellulose preparation, liberating soluble 14C-labelled compounds.

The catabolism of phosphatidylethanolamine by the rumen protozoon Entodinium caudatum and its conversion into the N-(1-carboxyethyl) derivative.

1. The N-(2-hydroxyethyl)alanine esterified to phosphatidic acid in anaerobic ciliate rumen protozoa has the l configuration. 2. Labelling experiments with Entodinium caudatum cultures using [(32)P]P(i) [2-(14)C]ethanolamine and (32)P- and (14)C-labelled phosphatidylethanolamine show that phosphatidylethanolamine is the direct lipid precursor of the N-(2-hydroxyethyl)alanine-containing phospholipid. 3. Labelling experiments with [(14)C]starch, [(14)C]lactate and [(14)C]pyruvate with E. caudatum cultures indicate that a three-carbon glycolytic intermediate is probably the precursor of the N-(1-carboxyethyl) grouping which substitutes on the amino group of phosphatidylethanolamine. 4. [(32)P]phosphatidylethanolamine is catabolized by E. caudatum forming initially glycerylphosphorylethanolamine and subsequently glycerophosphate and P(i). A little phosphorylethanolamine formed may possibly arise from bacterial enzymes ingested by the protozoa.

A study in the light and electron microscope of the extruded peristome and related structures of the rumen ciliate entodinium caudatum.

A study in the light and electron microscope of sections of the rumen ciliate Entodinium caudatum was undertaken in an attempt to elucidate the structure of the peristome when in the extruded state The conformation of the ciliary band on the peristome and the structure of the lining of the buccal cavity are described. The structure and position of a tuft of specialized sheet like extensions of the cell surface is also described.