Changes in metabolism and cell size of the anaerobic bacterium Selenomonas ruminantium 0078A at the onset of growth in continuous culture

Initial metabolism of Selenomonas ruminantium 0078A in continuous culture was characterized by a high lactate and low volatile fatty acid production; this was associated with poor growth as determined by bacterial dry weight production, yet individual cells were considerably larger than those of the inoculum. Biomass production increased, cell size decreased and the fermentation pattern reverted to the characteristic low lactate and high volatile fatty acid production after approximately 90 h growth.     

Effects of the ionophores monensin and tetronasin on simulated development of ruminal lactic acidosis in vitro

A continuous coculture of four ruminal bacteria, Megasphaera elsdenii, Selenomonas ruminantium, Streptococcus bovis, and Lactobacillus sp. strain LB17, was used to study the effects of the ionophores monensin and tetronasin on the changes in ruminal microbial ecology that occur during the onset of lactic acidosis. In control incubations, the system simulated the development of lactic acidosis in vivo, with an initial overgrowth of S. bovis when an excess of glucose was added to the fermentor. Lactobacillus sp. strain LB17 subsequently became dominant as pH fell and lactate concentration rose. Both ionophores were able to prevent the accumulation of lactic acid and maintain a healthy non-lactate-producing bacterial population when added at the same time as an excess of glucose. Tetronasin was more potent in this respect than monensin. When tetronasin was added to the culture 24 h after glucose, the proliferation of lactobacilli was reversed and a non-lactate-producing bacterial population developed, with an associated drop in lactate concentration in the fermentor. Rises in culture pH and volatile fatty acid concentrations accompanied these changes. Monensin was unable to suppress the growth of lactobacilli; therefore, in contrast to tetronasin, monensin added 24 h after the addition of glucose failed to reverse the acidosis. Numbers of lactobacilli and lactate concentrations remained high, whereas pH and volatile fatty acid concentrations were low.     

Effects of the ionophores monensin and tetronasin on simulated development of ruminal lactic acidosis in vitro.

A continuous coculture of four ruminal bacteria, Megasphaera elsdenii, Selenomonas ruminantium, Streptococcus bovis, and Lactobacillus sp. strain LB17, was used to study the effects of the ionophores monensin and tetronasin on the changes in ruminal microbial ecology that occur during the onset of lactic acidosis. In control incubations, the system simulated the development of lactic acidosis in vivo, with an initial overgrowth of S. bovis when an excess of glucose was added to the fermentor. Lactobacillus sp. strain LB17 subsequently became dominant as pH fell and lactate concentration rose. Both ionophores were able to prevent the accumulation of lactic acid and maintain a healthy non-lactate-producing bacterial population when added at the same time as an excess of glucose. Tetronasin was more potent in this respect than monensin. When tetronasin was added to the culture 24 h after glucose, the proliferation of lactobacilli was reversed and a non-lactate-producing bacterial population developed, with an associated drop in lactate concentration in the fermentor. Rises in culture pH and volatile fatty acid concentrations accompanied these changes. Monensin was unable to suppress the growth of lactobacilli; therefore, in contrast to tetronasin, monensin added 24 h after the addition of glucose failed to reverse the acidosis. Numbers of lactobacilli and lactate concentrations remained high, whereas pH and volatile fatty acid concentrations were low.     

Control of lactate production by Selenomonas ruminantium: homotropic activation of lactate dehydrogenase by pyruvate.

Selenomonas ruminantium produced one mole of D(-)-lactate per mole of glucose used at all dilution rates in ammonia-limited continuous culture. In contrast, lactate production varied according to the dilution rate when glucose was the limiting nutrient. At dilution rates of less than 0.2 h-1, acetate and propionate were the main fermentation products and lactate production was low. At dilution rates above 0.2 h-1, the pattern changed to one of high lactate production similar to that under ammonia limitation. Experiments with cell-free extracts of S. ruminantium showed that D(-)-lactate dehydrogenase had sigmoidal kinetics consistent with homotropic activation of the enzyme by its substrate, pyruvate. This feature allows S. ruminantium to amplify the effects of relatively small changes in the intracellular concentration of pyruvate to cause much larger changes in the rate of production of lactate. Some confirmation that this mechanism of control occurs under physiological conditions was obtained in glucose-limited culture, in which the sigmoidal increase in lactate production was accompanied by a linear increase in pyruvate excretion as the dilution rate increased.     

Effect of Dicarboxylic Acids and Aspergillus oryzae Fermentation Extract on Lactate Uptake by the Ruminal Bacterium Selenomonas ruminantium

The objective of this study was to determine the effects of l-aspartate, fumarate, l-malate, and an Aspergillus oryzae fermentation extract (Amaferm) on growth on lactate as well as lactate uptake by Selenomonas ruminantium HD4. Growth of S. ruminantium in medium that contained 2 g of dl-lactate per liter was stimulated approximately twofold by 10 mM l-aspartate, fumarate, or l-malate after 24 h. Both l-aspartate and fumarate increased lactate uptake over 4-fold, while l-malate stimulated uptake over 10-fold. Amaferm enhanced lactate uptake at all concentrations tested (0.5 to 50 g/liter), and the 10-g/liter level increased uptake over 12-fold. A filter-sterilized Amaferm filtrate increased lactate uptake over sevenfold, and growth on lactate was stimulated over twofold by either 2 or 5% (vol/vol) Amaferm filtrate. The Amaferm filtrate also increased the production of acetate, propionate, total volatile fatty acids, and Y(lactate) from lactate-grown cells. Since the increase in propionate production was greater relative to acetate, a decrease in the acetate:propionate ratio was observed. The concentration of l-malate in the Amaferm filtrate was 1.45 mM, and it appeared that the l-malate content of Amaferm played a role in the stimulation of growth on lactate as well as lactate uptake by S. ruminantium treated with Amaferm.     

Ruminal Fermentation and Sugar Concentrations

The maximal amounts of growth of Selenomonas ruminantium were examined in the media containing various amounts of glucose. The yields of cells per unit weight of glucose are linear functions to glucose concentrations in the ranges between zero to 0.005% and 0.005 to 0.7%, Cell yields per glucose are greater in the former range, indicating greater a-mounts of energy are available per glucose at lower concentrations. Growth responses in lactate media containing various amounts of glucose showed that the preincubation with larger amounts of glucose is inhibitory for the following growth and metabolism of lactate. The organism produces predominantly lactate in the glucose medium. However, volatile fatty acid productions increase when the initial concentrations of glucose become low. Isotopic studies showed that the lactate utilization yielding volatile fatty acids is inhibited by the preceding metabolism of high concentrations of glucose. These results were discussed with regard to normal and abnormal fermentations in the rumen.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

一株瘤胃源乳酸利用菌的分离鉴定及其体外代谢特性

【目的】从饲喂高精料的本地山羊瘤胃内分离到一株利用乳酸并能产生大量丙酸的菌株L9,并进一步研究了该菌在调控瘤胃微生物发酵中的作用。【方法】采用厌氧培养技术,结合形态、生理生化特性和16SrRNA基因序列分析结果。【结果】该菌株被鉴定为反刍兽新月形单胞菌（Selenomonas ruminantium）。该菌株体外代谢特性研究表明,L9可利用乳酸作为唯一碳源,该菌在24h内可对90mmol／L的乳酸完全降解。体外摸拟瘤胃急性酸中毒的发酵试验结果表明,以淀粉为底物时,与对照组相比,添加菌株L9可显著降低瘤胃微生物体外培养体系中乳酸浓度,提高pH值,提高总挥发性脂肪酸和丙酸浓度,并显著降低乙酸与丙酸的浓度比（P〈0．05）。【结论】结果显示,菌株L9是一株可代谢乳酸,促进丙酸生成,提高总挥发性脂肪酸浓度的有益瘤胃细菌。     

Metabolism of the rumen bacterium Selenomonas ruminantium grown in continuous culture

Selenomonas ruminantium 0078A was grown in a glucose-limited chemostat over a dilution rate range of 0.049-0-137/h. Fermentation products were acetate, propionate, succinate, lactate and C0₂; traces of ethanol were also detected. Succinate accounted for up to 52% of the substrate glucose carbon. When dilution rate was increased without a concomitant increase in glucose supply per unit time there were changes in the fermentation pattern which were not apparent when both dilution rate and glucose supply were simultaneously increased; the molar proportion of acetate increased at the expense of propionate.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.     

Isolation, Culture, and Fermentation Characteristics of Selenomonas ruminantium var. bryanti var. 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.     

Nutritional Requirements of Selenomonas ruminantium for Growth on Lactate, Glycerol, or Glucose

The nutritional requirements of Selenomonas ruminantium HD4 for growth on glucose, glycerol, or lactate were investigated to clarify the results of previous studies and to relate the nutrition of the organism to its physiology. The organism required l-aspartate, CO(2), p-aminobenzoic acid, and biotin for growth on a lactate-salts medium that contained small amounts of dithiothreitol. Aspartate could be replaced by l-malate or fumarate but not by succinate or l-asparagine. Requirements for growth with glycerol as an energy source were similar, except that aspartate was not required. With glucose as the energy source, neither aspartate nor p-aminobenzoic acid was required, but a requirement for volatile fatty acids, which could be met by n-valerate, was observed. CO(2) was required for growth on lactate or glycerol but not on glucose on complex media containing Trypticase and yeast extract. Sulfide could be used as the sole source of sulfur.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria.

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactate by Selenomonas ruminantium.

A method is described for increasing the production of H2 from glucose or lactate by Selenomonas ruminantium by sequential transfers in media containing pregrown Methanobacterium ruminantium. The methanogen uses the H2 formed by the selenomonad to reduce CO2 to CH4. Analysis of fermentation products from glucose showed that lactate was the major product formed from glucose by S. ruminantium alone. Several sequential transfers in the presence of the methanogen caused a marked decrease in lactate production, which was accompanied by an increase in acetate. When lactate was the fermentation substrate, S. ruminantium alone produced propionate, acetate, and CO2. Addition to the pregrown methanogen in the sequential transfer procedure caused a significant decrease in the production of propionate and an increase in acetate formed from lactate. These results are interpreted in terms of the influence of H2 utilization by the methanogen on the production of H2 versus lactate or propionate from reduced pyridine nucleotides by S. ruminantium.     

Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactate by Selenomonas ruminantium

A method is described for increasing the production of H2 from glucose or lactate by Selenomonas ruminantium by sequential transfers in media containing pregrown Methanobacterium ruminantium. The methanogen uses the H2 formed by the selenomonad to reduce CO2 to CH4. Analysis of fermentation products from glucose showed that lactate was the major product formed from glucose by S. ruminantium alone. Several sequential transfers in the presence of the methanogen caused a marked decrease in lactate production, which was accompanied by an increase in acetate. When lactate was the fermentation substrate, S. ruminantium alone produced propionate, acetate, and CO2. Addition to the pregrown methanogen in the sequential transfer procedure caused a significant decrease in the production of propionate and an increase in acetate formed from lactate. These results are interpreted in terms of the influence of H2 utilization by the methanogen on the production of H2 versus lactate or propionate from reduced pyridine nucleotides by S. ruminantium.     

Molecular dissection of the Selenomonas ruminantium cell envelope and lysine decarboxylase involved in the biosynthesis of a polyamine covalently linked to the cell wall peptidoglycan layer

The wild type of Selenomonas ruminantium subsp. lactilytica, which is a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, requires one of the normal saturated volatile fatty acids with 3 to 10 carbon atoms for its growth in a glucose medium; however, no such obligate requirement of fatty acid is observed when the cells are grown in a lactate medium. This bacterium is characterized by a unique structure of the cell envelope and a novel lysine decarboxylase and its regulatory protein. In the first part of this article, we will refer to the chemical structure of phospholipid and lipopolysaccharide in the cell membranes of this bacterium compared with that from the general Gram-negative bacteria for understanding their biological functions. S. ruminantium has neither free nor bound forms of Braun lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. However, S. ruminantium has cadaverine, which links covalently to the peptidoglycan as a pivotal constituent for the cell division. In the second part of this article, we will refer to the chemical structure of the cadaverine-containing peptidoglycan, its biosynthesis, and the biological function. In the third part of this article, we will depict the molecular cloning of the genes encoding S. ruminanitum lysine decarboxylase (LDC) and its regulatory protein of 22-kDa (22-kDa protein; P22) which has similar characteristics to that of antizyme of ornithine decarboxylase in eukaryotic cells, and the molecular dissection of these proteins for understanding the regulation of cadaverine biosynthesis. Finally, we will illustrate a proposed structure of the cell envelope, a processes of biosynthesis of the cadaverine-containing peptidoglycan layer, and the LDC degradation mechanism in S. ruminantium, on the basis of the analyses of the cell envelope components, the results from the in vitro experiments on the biosynthesis of the peptidoglycan layer, and the current status of the knowledge on LDC and P22 in this organism.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.