The hydrogenosomal enzyme hydrogenase from the anaerobic fungus Neocallimastix sp. L2 is recognized by antibodies, directed against the C-terminal microbody protein targeting signal SKL.

The question was addressed whether antibodies directed against the general microbody C-terminal protein targeting signal SKL recognized hydrogenosomal proteins from Neocallimastix sp. L2. Immunofluorescence, immunocytochemistry and Western blotting experiments using these antibodies indicated the presence of hydrogenosomal proteins containing SKL-COOH. One of these proteins, the hydrogenase, was purified to homogeneity. It has a native molecular mass of 87 kDa and consists of two subunits of approximately 30 and 60 kDa, both cross-reacting with anti-SKL antibodies. Its activity could be inhibited by CO, NO2-, and acetylene, suggesting a (Ni-Fe-Se) hydrogenase. Immunocytochemistry using polyclonal antibodies raised against the hydrogenase revealed the location of this protein in the hydrogenosomal matrix. The results described in this paper suggest that hydrogenosomes from Neocallimastix sp. L2 are related to microbodies from aerobic eukaryotes and support the idea of a common evolutionary origin for these organelles.

Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome.

The presence of hydrogenosomes in phylogenetically distinct anaerobic eukaryotes implies that they have been acquired independently, and previously reported differences in ultrastructure among taxa have suggested that some hydrogenosomes have different origins. Of particular interest are reports that Neocallimastix frontalis hydrogenosomes resemble microbodies in possessing a single membrane, in contrast to those in ciliates and trichomonads which have two and thus resemble mitochondria. In this investigation we have clearly demonstrated that N. frontalis hydrogenosomes possess two, rather than one, closely apposed membranes and in some preparations cristae-like structures were observed. These observations have led us to reject the microbody hypothesis and provide some indirect support for a possible mitochondrion origin as proposed for other hydrogenosomes. N. frontalis hydrogenosomes were shown to lack an associated genome as previously demonstrated for trichomonad hydrogenosomes. This might be explained by assuming that a mitochondrial genome encoding proteins for aerobic function is no longer necessary for either organelle.

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria.

From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.

Role of DL-lactic acid as an intermediate in rumen metabolism of dairy cows.

The role of DL-lactic acid as an intermediate in the rumen of a Friesian X Holstein dairy cow adapted to a diet of hay ad libitum plus 12 kg of a concentrate mixture was studied in vitro and in vivo. Concentrations of soluble sugars in the rumen fluid became maximal at 30 min postfeeding, but at 90 min no sugars were detectable. The DL-lactate concentration increased very rapidly to about 30 mm at 30 min after feeding, whereas the maximum total VFA concentration was reached 15 min later. More than 80% of the DL-lactate fermented to VFA was converted by Megasphaera elsdenii. Whereas only 16% of L-lactate was fermented to propionate, 75% of the D-lactate was converted to propionic acid. When all soluble sugars had been fermented, the participation of M. elsdenii to lactate fermentation declined and fermentation patterns for D- and L-lactate became similar yielding mostly acetate. Except for a brief period immediately after feeding, DL-lactate did not appear to be an important precursor of VFA in the rumen of a cow adapted to concentrate feeding. DL-lactate may become a more important intermediate in rumen fermentation temporarily when dairy cows are gradually changed from a hay diet to a diet including concentrates. The first 30 d after parturition, when the changeover takes place, is an unstable period, during which the microbial population is changing to fit the new environment.

An analysis of the buffer system in the rumen of dairy cattle.

A method is presented for the analysis of buffer systems in the rumen using the first derivation of titration curves. Bicarbonate and volatile fatty acids (VFA) are the main components of the buffering system in the rumen fluid of dairy cattle under widely different feeding conditions. Phosphate from saliva is of little importance as a buffer, but neutralizes acids produced in the rumen. After studying five cows during the peripartal period a spontaneous and transient increase in the concentrations of VFA and a soluble marker (PEG) as well as a drop in pH and in the bicarbonate concentrations not related to feeding was observed in two animals that were sampled several hours before parturition. The potential risk of provoking rumen disturbances upon feeding animals close to the time of parturition, when buffering capacity may be minimal, is stressed.

Association of germfree mice with intestinal microfloras obtained from "normal" mice.

A cultured microflora obtained from the caecum of a "normal" mouse was given to 4 groups of germfree mice and was supplied 1x, 2x, 3x and 4x respectively at 5-day intervals. Another group received a 10(-7) dilution of the caecal flora while a group associated with an 'SPF' flora served as control. The difference (measured by 8 parameters) between mice supplied with the cultured flora or with a 10(-7) dilution, both given once only, was small. Supplying the flora 3x resulted in more 'normal' mice compared with mice which received the flora once or twice. The caeca of specified-pathogen-free mice contained more bacteria per gram (microscopic bacterial count), less aerobic and anaerobic bacteria per gram (viable counts), while the yield as percentage of the microscopic bacterial count was lower as compared with the group to which a cultured flora was supplied 4 times.

'Normalization' of germfree mice with anaerobically cultured caecal flora of 'normal' mice.

Germfree (GF) mice were inoculated with a cultured flora from 10(-1), 10(-3), 10(-5), and 10(-7) dilutions of caecal contents from a 'normal' mouse. GF mice associated with a flora of a 'normal' mouse served as controls. The following intestinal parameters were determined: Colonization resistance (CR), Relative caecal weight (RCW), villus:crypt ratio (jejunum and ileum), IgA-producing cells (jejunum and ileum), beta-aspartyl glycine (faeces), volatile and non-volatile fatty acids (caecum) and bile acids (faeces). Only the 10(-1) culture was able to induce similar changes in the GF mice to a 'normal' flora. The GF + 10(-5) and GF + 10(-7) groups deviated markedly from the controls while the GF + 10(-3) group showed in general intermediate values between GF + SPF and GF + 10(-1) on the one hand and GF + 10(-5) and GF + 10(-7) on the other hand. beta-aspartyl glycine was present only in the GF + 10(-7) group. Scanning electron microscopy (SEM) of ileal contents revealed segmented filamentous organisms in the ileum of controls and the GF + 10(-1) group. The faecal flora consisted mainly of fusiform organisms. In the faeces of the 10(-5) and 10(-7) groups increasing amounts of non-bacterial matter were found, while in the faeces of the other groups virtually only bacteria were seen.

Regulation of lactate metabolism in the rumen.

The regulation of lactic acid production, the regulation of lactate fermentation and the role of lactate as intermediate in the rumen metabolism was studied. The pH had a pronounced effect on all three processes and therefore buffer capacity of the rumen contents is also described. Starch gave much less rise to lactic acidosis than soluble sugars, as glucose and fructose. Most bacteria grow faster and therefore produce more lactic acid when amino acids and/or soluble proteins are present in the diet. Activity of LDH (lactate dehydrogenase) of mixed rumen microorganisms is regulated by the NADH/NAD(H) balance and the ATP concentration. About 60% of the LDH in mixed rumen microorganisms is fructose-1, 6-diphosphate independent. Megasphaera elsdenii ferments 60 to 80% of the lactate fermented in the rumen of dairy cattle. Lactate accumulates only when the glycolytic flux (hexose units fermented per unit time per microorganism) is high. During adaptation, the glycolytic flux is increased and lactate may accumulate. After adaptation to a certain diet, the number of microorganisms is changed and the glycolytic flux again is normal and lactate is only a minor intermediate in rumen metabolism.

[Ruminants as breeding animals (author's transl)]. / De herkauwer also veredelingsdier.

Ruminants can be considered as up graders of the quality of food when they are kept on feeds low in nitrogen and rich in structural carbohydrates such as cellulose. Many feeds and agricultural wastes of no value or unattractive to monogastric animals can be converted into tractive power, milk, wool, meat and offspring by ruminants. To attain levels of production, concentrates which are readily degraded and high in protein are being increasingly fed, which results in improper use of the ruminant, production losses and metabolic disorders due to unstable rumen fermentation. This is also associated with the fact that our domesticated cattle belong to a group of ruminants particularly equipped to deal with roughage. Most other ruminants in the group of the large roughage feeders are even superior to European cattle breeds in digesting poor roughage. When it would become necessary to increase the use of roughages and agricultural lignocellulose wastes in the near future, a large ruminal volume and a high salivation rate should be adopted as criteria of selection in breeding new cattle breeds.

[Degradation of cereal straw with lye]. / Ontsluiting van graanstro met loog.

Straw is a plentiful agricultural waste product. It cannot be used as the sole feed for ruminants and it cannot even serve to satisfy maintenance requirements. The rumen microbes are not capable of breaking down the cell walls in cereal straw rapidly enough or to a sufficient extent. To improve the utilization of straw, efforts were made to treat this material by physical, chemical and biological methods. Some of these procedures will only improve the intake of straw by the animal, whereas the primary object continues to be the improvement of digestibility. Alkali treatment of straw is the most suitable method for this purpose. The mode of action of alkali on the cell walls of straw is described in the present paper.

Isolation, culture, and fermentation characteristics of Selenomonas ruminantium var. bryantivar. n. from the rumen of sheep.

Large forms of Selenomonas sp. were isolated from the sheep rumen on a rumen fluid-glucose-agar medium by using a differential centrifugation technique to purify the inoculum. The cells from the six isolated strains were curved, gram-negative, strictly anaerobic crescents, and rapidly motile by flagella attached to the concave side of the cell. One or more of the volatile fatty acids were essential for growth. None of the strains produced indole or reduced nitrate. All strains grew on fructose, glucose, mannose, cellobiose, maltose, sucrose, and salicin. Fermentation end products from glucose were mainly lactate, acetate, propionate, and formate. Small amounts of succinate were formed. The final pH in a glucose medium ranged between 4.3 and 4.5. On the basis of the sugar fermentation characteristics and the capacity to form hydrogen sulfide from cysteine, it is suggested that one of the strains is a large form of Selenomonas ruminantium. The other five strains are designated S. ruminantium var. bryanti, var. n.

Reisolation of Ruminobacter parvum.

A cellulolytic gram-negative ovoid motile rod (strain GS III) was isolated from an in vitro incubation of ground barley straw in rumen fluid. The anaerobic, non-sporulating, mesophilic organism strongly answered the original description of Ruminobacter parvum (Kaars Sijpesteijn, 1948). The strain GS III fermented pyruvate, D-arabinose, D-xylose, cellobiose, sucrose, maltose, cellulose, dextrin, xylan and pectin. Products from cellobiose were D-lactate, ethanol, acetate, hydrogen and carbon dioxide. A heat-resistant factor in yeast extract that was not a B-vitamin, a metal or one of the volatile fatty acids was required for growth. The mol % G + C was 51.9. The organism was lost after prolonged subculture.

Thermophiles: A life at elevated temperatures.

Interest in the ecology, physiology and evolution of microorganisms adapted to growth at relatively high temperatures (up to 110°C) has increased enormously during the past two decades. This interest was stimulated by the discovery of marine hydrothermal vent ecosystems, and also by awareness of the potential of thermophilic microbes in biotechnological processes. Subsequent attempts to isolate new thermophilic organisms have been very successful. Moreover, these results, in combination with much-improved techniques for studying the phylogeny of microorganisms, have renewed interest in the evolution of microbes and the early development of life.

Calculation of k and v from substrate concentration versus time plot.

A simple method for the calculation of kinetic parameters (K(m), V(max)) under conditions of changing substrate concentrations is presented. An application of the method to detect shifts in groups involved in the utilization of a substrate in a mixed microbial culture is given.

On the contribution of the acrylate pathway to the formation of propionate from lactate in the rumen of cattle.

The effect of chloral hydrate, an inhibitor of methanogenesis, on the participation of the acrylate pathway in the formation of propionate from lactate in rumen contents of cattle was studied in vitro. Addition of chloral hydrate resulted in only a small stimulation of the acrylate pathway, much lower than the stimulation of propionate production by chloral hydrate. This means that the flux of carbon through both the acrylate and the dicarboxylic acid pathway is increased during chloral hydrate feeding. The influence of time of sampling after feeding on the contribution of the acrylate pathway was studied in a separate experiment. A marked drop in the participation of the acrylate pathway in propionate formation from lactate during at least 2 h after feeding was observed, whereafter a rapid rise to prefeeding levels occurred.

Utilization of organic nitrogen sources by two phytoplankton species and a bacterial isolate in pure and mixed cultures.

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.