Continuous cultivation of rumen microorganisms,a system with possible application to the anaerobic degradation of lignocellulosic waste materials

Summary An in vitro continuous fermentation device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liquids found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixture was used as a solid growth substrate. During a test period of 65 days the artificial rumen fermenter showed stable operation with respect to ciliate numbers, fibre degradation and volatile fatty acids production. Values obtained were comparable to those found in vivo. Optimal fibre degradation and volatile fatty acids production were maintained when hydraulic retention times (HRT) ranged from 11 to 14 h. At these HRT-values ciliate numbers were maintained at about 8.5×10⁴ cells per ml. Ciliate numbers declined drastically at HRT-values above 14h. A fermenter inoculated with a small volume of rumen fluid (1:100, v/v) reached normal protozoal numbers, fibre degradation and volatile fatty acids productions after a start up period of only 8 to 10 days. The possible application of rumen microorganisms for an efficient degradation of lignocellulosic waste material in an artificial rumen digester is discussed.     

Isolation and identification of 5,10-methenyl-5,6,7,8-tetrahydromethanopterin, a coenzyme involved in methanogenesis

Abstract The structure of the coenzyme involved in methanogenesis, which was known previously as 'Yellow fluorescent compound' or carboxy-5,6,7,8-tetrahydromethanopterin, has been elucidated by means of nuclear magnetic resonance and UV spectroscopy. The compound is now identified as 5,10-methenyl-5,6,7,8-tetrahydromethanopterin.     

Quantitation of coenzyme F420 in methanogenic sludge by the use of reversed-phase high-performance liquid chromatography and a fluorescence detector

Summary With the use of acetone extraction, reversed-phase High-Performance Liquid Chromatography and fluorimetric monitoring, the quantity of coenzyme F₄₂₀ in mixed liquors and rumen contents can be measured. A synthetic analog of coenzyme F₄₂₀ is used as an internal standard to compensate for differences in fluorimetric monitoring. The method allows the detection of one picomol of coenzyme F₄₂₀ and the differentiation between different forms of the coenzyme known to be present in various methanogenic bacteria.     

Interconnection of methanogenic and acetogenic pathways

Abstract Methanopterin displays a folate-like biochemistry: methenyl-, methylene- and methyltetrahydromethanopterin are intermediates in the process of methanogenesis. A corrinoid-enzyme is involved in methanol conversion and the possible function of such enzymes in cell-carbon synthesis and acetate conversion is discussed. Energy conservation is proposed to proceed via the establishment of a proton motive force through a vectorial reduction in the final step of methanogenesis and via substrate level phosphorylation in the oxidation of formyltetrahydromethanopterin. The biochemical reactions of methanogenesis and acetogenesis are compared. Methanogenic bacteria appear to use an aberrant set of coenzymes in almost all of the reactions involved in methanogenesis.     

Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5-nitrosalicylaldehyde

Summary The histochemical fluorescence method using 5-nitrosalicylaldehyde for the demonstration of cathepsin B activity has been used. Precipitation of the fluorescent final reaction product was analysed continuously during incubation for cathepsin B activity. Unfixed cultured human fibroblasts as well as cryostat sections of mouse metacarpal bone explants were used. Continuous monitoring of the formation of the fluorescent reaction product showed that after a certain lag phase, depending on the enzyme activity in the tissue, discrete granules appeared which became increasingly fluorescent with incubation time. Subsequently, recrystallization and redistribution of the final reaction product started to occur. It is concluded that the coupling reaction with 5-nitrosalicylaldehyde is sufficiently fast for a proper localization of proteinase activity and can be used for kinetic analysis of enzyme activity. The method provides indications of relative amounts of cathepsin B activity in different cell types within a tissue section. It appeared from the study on metacarpal bone explants that fibroblasts in perichondrium and periosteum contained a relatively high cathepsin B activity whereas chondrocytes showed a low but distinct activity. This observation suggests that cysteine proteinases are not only involved in collagen degradation by fibroblasts but that they also play a role in the intracellular digestion of collagen by chondrocytes.     

Allantoin racemase: a new enzyme from Pseudomonas species.

1. Allantoin racemase is a novel enzyme which catalyzes the conversion of S(+)-and R(minus)-allantoin into the racemate. 2. The enzyme is present in Pseudomonas testosteroni, Pseudomonas putida and five biotypes of Pseudomonas fluorescens, but absent in a number of other Pseudomonas species. 3. The enzyme of Ps. testosteroni was purified 133-fold and exposes optimal activity at pH 8.0-8.2 and 50 degrees C. The enzyme is stable on heating for 15 min at 70 degrees C. 4. The enzyme appeared to be specific for the optical isomers of allantoin and no cofactors are involved in the reaction. 5. The optical aspecificity of allantoinase of Proteus rettgeri was reaffirmed.     

Biochemical and ultrastructural changes in Staphylococcus aureus treated with staphylococcin 1580

Incorporation of precursors into macromolecules is immediately arrested upon treatment of Staphylococcus aureus cells with staphylococcin 1580. Except for a degradation of RNA, induced after about 40 min, no degradation of macromolecules is observed, and no trichloroacetic acid-insoluble components are released from the cells.The protein composition and content of membranes are not affected by staphylococcin 1580 treatment. The fatty acid pattern of cells is not significantly altered.Protoplasts do not lyse apparently upon treatment with staphylococcin 1580, but undergo morphological alterations.Thin sections of cells treated with the bacteriocin for 30 min show extensive mesosome-like structures, mostly arranged in honeycomb arrays connected to the plasma membrane, and alterations in the nucleoid area. Freeze-etched preparations taken after that time reveal alterations in the plasma membrane, presumably in relation to the formation of the mesosomal structures. No alterations were observed after bacteriocin treatment for 5 min, although at that time the permeability of the membrane is strongly affected.The implications of the observed changes with the development of irreversible lesions in the cells are discussed.     

Purification and characterization of a complex-bound and a free β-1,4-endoxylanase from the culture fluid of the anaerobic fungus Piromyces sp. strain E2

Two extracellular xylanases were purified to homogeneity from the culture filtrate of the anaerobic fungus Piromyces sp. strain E2 and their properties were studied. The enzymes are present in a High Molecular Mass complex (HMM-complex) and as free protein in nearly equal amounts. Both enzymes are most likely identical as all biochemical characteristics were identical. The molecular masses of the enzymes are 12.5 kDa, as estimated by gel chromatography and electrophoretic mobility. The activities of both enzymes are optimal at pH 6.0 and 50°C and the enzymes are stable up to 72h at 40°C. The enzymes have a pI of 9.1. The K _m and V _max, determined with xylan from oat spelts, were 3 mg · ml^-1 and 2600 IU · mg^-1 protein. The enzymes are active both on soluble and insoluble oat spelt xylan. The purified xylanases are inactive against Avicel, carboxymethylcellulose, p-nitrophenyl--d-glucoside, and p-nitrophenyl--d-xyloside. The products of the pure enzymes are predominantly xylo-oligosaccharides, indicating that the enzymes act as endoxylanases (1,4--d-xylan xylanohydrolases, EC 3.2.1.8).     

Effect of fixation on activity and cytochemistry of hydrogenosomal enzymes in Trichomonas vaginalis.

The effect of fixation on the activity of malate dehydrogenase (decarboxylating) and pyruvate synthase was investigated in Trichomonas vaginalis. Subsequently a cytochemical staining method was developed for the demonstration of malate dehydrogenase activity in hydrogenosomes. After fixation of cells in low concentrations of glutaraldehyde and incubation in the presence of malate and the tetrazolium compound 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. During the whole procedure strictly anaerobic conditions were required. Attempts to develop an analogous procedure for pyruvate synthase failed because even low concentrations of glutaraldehyde strongly inhibited enzyme activity. When cells were fixed in low concentrations of glycolaldehyde and acetaldehyde, a high enzyme activity was retained, but no staining could be achieved. Application of both staining methods to the sapropelic ciliates Trimyema compressum and Plagiopyla nasuta gave negative results.