Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

The role of the rumen ciliate protozoa for methane suppression caused by coconut oil

Diets containing either coconut oil or rumen-protected fat (54 g kg⁻¹ dry matter each) were supplied to Rumen Simulation Technique fermenters filled with faunated and defaunated rumen fluid in a 2 × 2 factorial design. Defaunation immediately reduced methane formation by about 40% with each diet. With coconut oil, methane gradually declined in faunated and defaunated rumen fluid. Finally, the extent of methane suppression was similar, both with coconut oil and with defaunation. Independently of the status of protozoa, the population of methanogens in rumen fluid was significantly reduced by coconut oil. The results suggest that defaunation and coconut oil independently and additively suppress rumen methanogenesis.     

Association of methanogenic bacteria with rumen protozoa

Methanogenic bacteria superficially associated with rumen entodiniomorphid protozoa were observed by fluorescence microscopy. A protozoal suspension separated from strained rumen fluid (SRF) by gravity sedimentation exhibited a rate of methane production six times greater (per millilitre) than SRF. The number of protozoa (per millilitre) in the protozoal suspension was three times greater than that of SRF; however, the urease activity of this fraction was half that of SRF. The methanogenic activity of SRF and the discrete fractions obtained by sedimentation of protozoa correlated with the numbers of protozoa per millilitre in each fraction. Gravity-sedimented protozoa, washed four times with cell-free rumen fluid, retained 67-71% of the recoverable methanogenic activity. Thus it is evident from our observations that many methanogens adhere to protozoa and that the protozoa support methanogenic activity of the attached methanogens. When protozoa-free sheep were inoculated with rumen contents containing a complex population of protozoa, methanogenic activity of the microflora in SRF samples was not significantly enhanced.     

2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.     

The cryopreservation of some large ciliate entodiniomorphid protozoa taken from the rumen

M.H.P. FUNGARO, M.L.C. VIEIRA, A.A. PIZZIRANI-KLEINER AND J.L. DE AZEVEDO. 1996. Random amplified polymorphic DNA (RAPD) was used in order to analyse the relationships among 13 isolates of Metarhizium anisopliae var. anisopliae . Six of them were isolated from Deois flavopicta (Stal) (Hemiptera—Homoptera: Cercopidae) in different regions of Brazil. The other seven were isolated from soil in Paraná State in Southern Brazil. The isolates were grouped by cluster analysis using Dice similarity index. The results show that isolates of M. anisopliae var. anisopliae are extremely diverse (47% similarity) but those isolated from D. flavopicta present only a moderate degree of variation (82% similarity) when compared with the wide diversity (31% similarity) found in the group isolated from soil. These results suggest that M. anisopliae var. anisopliae has developed host specificity.     

2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa.

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.     

Interactions between the methanogen Methanosarcina barkeri and rumen holotrich ciliate protozoa

Interspecies hydrogen transfer between rumen holotrich ciliate protoza and methanogenic bacteria has been demonstrated. As a result of the metabolic interaction with Methanosarcina barkeri , the metabolite profile of Isotricha spp. was altered and the production of butyrate and lactate was suppressed in the presence of the methanogen.
Use of membrane-inlet mass spectrometry confirmed that the presence of rumen holotrich ciliates reduced the apparent sensitivity of methanogenesis to the inhibitory effects of oxygen; a gas phase concentration of 7·4 kPa oxygen was required to inhibit methanogenesis in the presence of protozoa, while in pure cultures of M. barkeri , methanogenesis was inhibited by a gas phase oxygen concentration of 1·0 kPa.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

The influence of headspace gas composition on the production of VFA by rumen ciliate protozoa

Aerobic bacterial growth was assessed in a simple rectangular vessel with good mixing achieved solely by air flow. Growth became oxygen limited at culture densities below 1·5 mg dry wt/ml, even at maximum air flow and the extent of oxygen-sufficient growth depended on the volume of the culture and the air flow rate. However, the system permitted high retention of biomass growing attached to kiesel-ghur beads, indicating conditions of low shear. The system may be suitable for cheap cultivation of slow-growing micro-organisms and for anaerobic or shear-sensitive cultures.     

Subcellular distribution of polysaccharide depolymerase and glycoside hydrolase enzymes in rumen ciliate protozoa

The distribution of polysaccharide depolymerase and glycoside (acid) hydrolase activity in nine genera of rumen entodiniomorphid and holotrich ciliate protozoa was examined by differential centrifugation. Sedimentable activity was detected in all of the protozoa examined and occurred principally in fractions that were prepared by centrifugation at 1000g for 10 min, 10,000g for 10 min, and 20,000g for 20 min (fractions F1, F2, and F3). Acid phosphatase was present in these subcellular fractions which contained membrane-bound vesicles 0.1–0.8 m in size. The enzyme location profile of the subcellular fractions differed within the genera examined. The distribution of the enzyme activity in the subcellular fractions indicated the presence of distinct populations of hydrolase-containing organelles and other functional vesicles in the rumen ciliates.     

The effect of Aspergillus oryzae fermentation extract on the growth of fungi and ciliate protozoa in the rumen

When added to the diet of sheep, 2 g/d, Aspergillus oryzae fermentation extract (AO) stimulated total and cellulolytic bacterial numbers in rumen fluid by 34 and 90% respectively. AO had no effect on the numbers of protozoa or fungal zoospores. AO did not affect hydrogen production by the rumen fungi Neocallimastix frontalis (RE1), N. patriciarum (CX) or Piromonas communis (P) in pure culture or protozoal activity in vitro , estimated from the rate of breakdown of [¹⁴C] leucine-labelled Selenomonas ruminantium. It was concluded that increases in ruminal fibre digestion observed previously in animals fed AO, were most likely due to a stimulation of bacteria rather than eukaryotes in the rumen microbial population.     

Interactions between rumen bacteria and ciliate protozoa in their attachment to barley straw

The attachment of ¹⁴C-choline-labelled mixed rumen protozoa to barley straw in vitro was not significantly affected when bacteria prepared from rumen fluid were added to the incubation mixture. There was similarly little effect on protozoal attachment when the straw had already been colonized by a bacterial population for 24 h. In contrast, it was deduced from measurements of enzyme activities associated with straw that bacterial attachment was reduced if protozoa were present. Bacteria that had colonized the straw for 25 h beforehand were less susceptible to predation by protozoa.     

The effect of ciliate protozoa on the turnover of bacterial and fungal protien in the rumen of sheep

A four-fold increase (P < 0.001) in the total bacterial population in the rumen of sheep was observed following defaunation. A two-fold (P > 0.05) increase in the anacrobic fungal population was also observed. Defaunation caused a 10-fold (P < 0.001) reduction in the rate of protein turnover of Selenomonas rurninantium protein in vitro . However, only a two-fold reduction (P < 0.05) was observed in the rate of protein turnover of the rumen fungi Piromonas communis, Neocallimastix patriciarum and N. frontalis. It was concluded that although protozoa may play an important role in the turnover of fungal protein in the rumen, they have a greater role in the turnover of bacterial protein.