Can fungal zoospores be the source of energy for the rumen protozoa Eudiplodinium maggii?

Results of our earlier studies showed the ability of ciliates Eudiplodinium maggii to digest and metabolize commercial chitin. The natural source of this polysaccharide in the rumen are fungi. The objectives of present research were to determine the effect of fungal zoospores on the survival and population density of E. maggii to quantify the concentration of chitin in the cells of protozoa and to examine the ability of E. maggii, to ferment chitin of fungal zoospores. The cultivation experiment showed that the survival of protozoa was shorter than 4 days when the culture medium was composed of buffer solution and lyophilized fungal spores. An enrichment of this medium with wheat gluten prolonged the survival of ciliates up to 8 days. The supplementation of the last medium with meadow hay enabled the protozoa to survive for 28 days but a positive effect was observed only during the last 8 days of experiment. The chitin content was 0.27 ng and 0.21-0.35 ng per single zoospore and ciliate, respectively. An increase in the concentration of volatile fatty acids (VFA) was found when protozoa were incubated with zoospores. The production rate of VFA was 46.3 pM/protozoan per h whereas the endogenous production did not exceed 31 pM/protozoan per h. The molar proportion of acetic acid was 77.7% and these of butyric and propionic acids-12.2 and 11.0%, respectively. The obtained results make it evident that carbohydrates present in fungal zoospores were utilized by protozoa in energy yielding processes.

ß-Fructofuranosidase and sucrose phosphorylase of rumen bacterium Pseudobutyrivibrio ruminis strain 3.

The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained ß-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7), while the bacteria maintained on sucrose showed only phosphorylase. Partially purified ß-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis-Menten kinetics with V(max) 5.64 µM/mg/min and K(m) 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis-Menten kinetics. The V(max) were 0.599 and 0.584 µM/mg/min, while K(m) were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The V(max) were, however, 1.37 and 1.023 µM/mg/min, while K(m) were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 µM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.

Can a fermentation gas mainly produced by rumen Isotrichidae ciliates be a potential source of biohydrogen and a fuel for a chemical fuel cell?

Bacteria, fungi and protozoa inhabiting the rumen, the largest chamber of the ruminants' stomach, release large quantities of hydrogen during the fermentation of carbohydrates. The hydrogen is used by coexisting methanogens to produce methane in energy-yielding processes. This work shows, for the first time, a fundamental possibility of using a hydrogen-rich fermentation gas produced by selected rumen ciliates to feed a low-temperature hydrogen fuel cell. A biohydrogen fuel cell (BHFC) was constructed consisting of (i) a bioreactor, in which a hydrogen-rich gas was produced from glucose by rumen ciliates, mainly of the Isotrichidae family, deprived of intra- and extracellular bacteria, methanogens, and fungi, and (ii) a chemical fuel cell of the polymer-electrolyte type (PEFC). The fuel cell was used as a tester of the technical applicability of the fermentation gas produced by the rumen ciliates for power generation. The average estimated hydrogen yield was ca. 1.15 mol H2 per mol of fermented glucose. The BHFC performance was equal to the performance of the PEFC running on pure hydrogen. No fuel cell poisoning effects were detected. A maximum power density of 1.66 kW/m2 (PEFC geometric area) was obtained at room temperature. The maximum volumetric power density was 128 W/m3 but the coulombic efficiency was only ca. 3.8%. The configuration of the bioreactor limited the continuous operation time of this BHFC to ca. 14 hours.

The utilization and digestion of cellulose by the rumen ciliate Diploplastron affine.

Rumen protozoa are known to contribute to fibre digestion, but the fibrolytic enzymes of the majority of ciliate species have been poorly recognised to date. The aims of the study were, first, to determine the influence of crystalline cellulose on the survival and population density of the ciliate Diploplastron affine when cultured in vitro, and second to identify and characterise the protozoal enzymes catalysing the hydrolysis of cellulose. It was found that crystalline cellulose, when added to a culture medium, increased the number of protozoa maintained in vitro. We observed that the bacteria-free ciliates fermented microcrystalline cellulose and produced 43.3 nmol volatile fatty acids/protozoon/h. A cell extract prepared from the bacteria-free ciliates degraded crystalline cellulose in the rate of 11.5 nmol released glucose/mg protein/min, whereas the degradation rates of carboxymethyl-cellulose (CMC), avicel and cellobiose were 343, 6.8 and 145 nmol released glucose/mg protein/min respectively. Two distinct peaks in the activity of relevant enzymes were identified following ion exchange chromatography of the protozoal cell extract and the presence of two different CMC-ases were confirmed by zymographic studies. CMC was mainly degraded to mono- and disaccharides but that some other oligosaccharides were also present. Cellobiose was the only product of avicel digestion.

Treponema zioleckii sp. nov., a novel fructan-utilizing species of rumen treponemes.

During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.

The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin.

BACKGROUND: The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I. RESULTS: The [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome. CONCLUSION: The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.

Effect of Entodinium caudatum on starch intake and glycogen formation by Eudiplodinium maggii in the rumen and reticulum.

This study aimed to quantify the engulfed starch and reserve α-glucans (glycogen) in the cells of the ciliates Eudiplodinium maggii, as well the α-glucans in defaunated and selectively faunated sheep. The content of starch inside the cell of ciliates varied from 21 to 183mg/g protozoal DM relative to the rumen fauna composition whereas, the glycogen fluctuated between 17 and 126mg/g dry matter (DM) of this ciliate species. Establishment of the population Entodinium caudatum in the rumen of sheep already faunated with E. maggii caused a drop in both types of quantified carbohydrates. The content of α-glucans in the rumen of defaunated sheep varied from 4.4 to 19.9mg/g DM and increased to 7.4-29.9 or 11.8-33.9mg/g DM of rumen contents in the presence of only E. maggii or E. maggii and E. caudatum, respectively. The lowest content of the carbohydrates was always found just before feeding and the highest at 4h thereafter. The α-glucans in the reticulum varied 7.5-40.1, 14.3-76.8 or 21.9-106.1mg/g DM of reticulum content for defaunated, monofaunated or bifaunated sheep, respectively. The results indicated that both ciliate species engulf starch granules and convert the digestion products to the glycogen, diminishing the pool of starch available for amylolytic bacteria.

Horizontal gene transfer from Bacteria to rumen Ciliates indicates adaptation to their anaerobic, carbohydrates-rich environment.

BACKGROUND: The horizontal transfer of expressed genes from Bacteria into Ciliates which live in close contact with each other in the rumen (the foregut of ruminants) was studied using ciliate Expressed Sequence Tags (ESTs). More than 4000 ESTs were sequenced from representatives of the two major groups of rumen Cilates: the order Entodiniomorphida (Entodinium simplex, Entodinium caudatum, Eudiplodinium maggii, Metadinium medium, Diploplastron affine, Polyplastron multivesiculatum and Epidinium ecaudatum) and the order Vestibuliferida, previously called Holotricha (Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium). RESULTS: A comparison of the sequences with the completely sequenced genomes of Eukaryotes and Prokaryotes, followed by large-scale construction and analysis of phylogenies, identified 148 ciliate genes that specifically cluster with genes from the Bacteria and Archaea. The phylogenetic clustering with bacterial genes, coupled with the absence of close relatives of these genes in the Ciliate Tetrahymena thermophila, indicates that they have been acquired via Horizontal Gene Transfer (HGT) after the colonization of the gut by the rumen Ciliates. CONCLUSION: Among the HGT candidates, we found an over-representation (>75%) of genes involved in metabolism, specifically in the catabolism of complex carbohydrates, a rich food source in the rumen. We propose that the acquisition of these genes has greatly facilitated the Ciliates' colonization of the rumen providing evidence for the role of HGT in the adaptation to new niches.

Methods for the cultivation of ciliated protozoa from the large intestine of horses.

This paper describes cultivation methods for ciliates from the digestive tract of horses. Members of three different genera were successfully grown in vitro for short periods of time. However, only cells belonging to the genus Blepharocorys, which resides in the horse's large intestine, were maintained for longer periods. This Blepharocorys culture was successfully grown in vitro after inoculation of freshly excreted horse faeces in culture medium containing a population of bacteria. The ciliates survived for over six months, and the density of their population varied between 1.7 × 10(3) and 2.4 × 10(3) cells mL(-1). Favourable conditions for the prolonged cultivation of this ciliate were observed when the medium was prepared by mixing horse faeces and 'caudatum' salt solution in a 1:1 V/V ratio together with food (60% powdered meadow hay, 16% wheat gluten, 12% barley flour and 12% microcrystalline cellulose) supplied as 0.20 mg mL(-1) culture per day.

Dynamic Buffer Capacity in Acid-Base Systems.

The generalized concept of 'dynamic' buffer capacity ßV is related to electrolytic systems of different complexity where acid-base equilibria are involved. The resulting formulas are presented in a uniform and consistent form. The detailed calculations are related to two Britton-Robinson buffers, taken as examples.

"Why Not Stoichiometry" versus "Stoichiometry­Why Not?" Part III: Extension of GATES/GEB on Complex Dynamic Redox Systems.

In the third part of a series of articles issued under a common title, some examples of complex dynamic redox systems are presented and considered from analytical and physico-chemical viewpoints; the analysis is a leitmotiv for detailed, physico-chemical considerations. All attainable physico-chemical knowledge is involved in algorithms applied for resolution of the systems, realized with use of iterative computer programs. The first redox system (System I) is related to titration of FeSO4 + H2C2O4 with KMnO4 solution in acidic (H2SO4) medium, where simultaneous determination of both analytes from a single curve of potentiometric titration is possible. The possibility of the formation of precipitates (FeC2O4 and/or MnC2O4) in this system is taken into considerations. The second system (System II) relates to the complete analytical procedure involved in the iodometric determination of Cu; four consecutive steps of this analysis are considered. As a reasonable tool for explanation of processes occurring during simulated redox titration, speciation diagrams are suggested. This explanation is based on graphical presentation of results obtained from the calculations. The calculations made for this purpose are performed in accordance with principles of the generalized approach to electrolytic systems (GATES) with generalized electron balance (GEB) or GATES/GEB and realized with use of iterative computer programs offered by MATLAB. The reactions proceeding in this system can be formulated, together with their efficiencies, at any stage of the titration. Stoichiometry is considered as the derivative concept when put in context with GATES/GEB. The article illustrates the enormous possibilities and advantages offered by GATES/GEB.

"Why not stoichiometry" versus "Stoichiometry--why not?" Part II: GATES in context with redox systems.

Redox equilibria and titration play an important role in chemical analysis, and the formulation of an accurate mathematical description is a challenge. This article is devoted to static and (mainly) dynamic redox systems; the dynamic systems are represented by redox titrations. An overview addresses earlier approaches to static redox systems (redox diagram plots, including Pourbaix diagrams) and to titration redox systems, thereby covering a gap in the literature. After this short review, the generalized approach to electrolytic systems (GATES) is introduced, with generalized electron balance (GEB) as its inherent part within GATES/GEB. Computer simulation, performed according to GATES/GEB, enables following the changes in potential and pH of the solution, together with chemical speciation at each step of a titration, thus providing better insight into this procedure. The undeniable advantages of GATES/GEB over earlier approaches are indicated. Formulation of GEB according to two approaches (I and II) is presented on the respective examples. A general criterion distinguishing between non-redox and redox systems is presented. It is indicated that the formulation of GEB according to Approach II does not need the knowledge of oxidation degrees of particular elements; knowledge of the composition, expressed by chemical formula of the species and its charge, is sufficient for this purpose. Approach I to GEB, known also as the "short" version of GEB, is applicable if oxidation degrees for all elements of the system are known beforehand. The roles of oxidants and reductants are not ascribed to particular components forming a system and to the species thus formed. This is the complete opposite of earlier approaches to redox titrations, based on the stoichiometric redox reaction, formulated for this purpose. GEB, perceived as a law of matter conservation, is fully compatible with other (charge and concentration) balances related to the system in question. The applicability of GATES/GEB in optimization a priori of chemical analyses made with use of redox titration is indicated. The article is illustrated with many examples of static and dynamic redox systems. The related plots are obtained from calculations made according to iterative computer programs. This way, GATES/GEB enables seeing details invisible in real experiments.