Stoichiometry of glucose and starch splitting by strains of amylolytic bacteria from the rumen and anaerobic digester

The stoichiometry of glucose and starch splitting by the amylolytic bacteria Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Eubacterium ruminantium and Clostridium sp. was followed. There were many differences in the ratios of metabolites and in growth yields, as well as in the cell composition, between the growth on glucose and starch. The bacteria employ different nutritional strategies with respect to both energy sources.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

The uptake and metabolism of bacteria, amino acids, glucose and starch by the spined and spineless forms of the rumen ciliate Entodinium caudatum

Spined and spineless forms of Entodinium caudatum were obtained by growth in vivo in the presence and absence, respectively, of Entodinium bursa. Washed suspensions of both forms engulfed all the bacteria tested although the spined form took them up 1.3 to 1.9 times more rapidly per unit volume of protozoon than did the spineless form. Buytrivibrio fibrisolvens and Selenomonas ruminantium were rapidly digested by the spined form after engulfment. Free amino acids were taken up on average 3.1 times and glucose approximately 60 times faster per unit volume of protozoon by the spined form. Limited amounts of protein were synthesized by the spined form from glucose and starch but engulfed bacteria and, to a lesser extent, free amino acids were probably the prinicpal sources of protein for growth of both forms.     

Isolation and characterization of Selenomonas ruminantium strains capable of 2-deoxyribose utilization.

Microbes from ruminal contents of cattle were selectively enriched by using 2-deoxyribose (2DR) as a substrate for growth. Bacterial isolates growing on 2DR were gram-negative, curved, motile rods. The isolates grew on a broad range of substrates, including deoxyribose, glucose, ribose, mannitol, and lactate as well as ribonucleosides and deoxyribonucleosides. The strains also grew on rhamnose (6-deoxymannose) but not DNA. Organic acids produced from growth on hexoses and pentoses included acetate, propionate, lactate, and succinate. The isolates were identified as Selenomonas ruminantium subsp. lactilytica on the basis of morphology, substrate specificity, and other biochemical characteristics. Several characterized species of ruminal bacteria were also screened for growth on 2DR, with only one strain (S. ruminantium PC-18) found able to grow on 2DR. Ethanol was produced by 2DR when strains were grown on ribose or 2DR.     

Some effects of uncouplers and inhibitors on growth and electron transport in rumen bacteria.

Uncouplers and inhibitors of electron transport affected growth and electron transport of rumen bacteria in various ways. Selenomonas ruminantium was not affected by inhibitor and uncoupler concentrations which affected growth and electron transport of Bacteroides ruminicola, B. succinogenes, and Butyrivibrio fibrisolvens. Inhibitors, when active, led to accumulation of reduced electron carriers before the site of action, but differences were found among organisms in the site of action of these inhibitors. Uncouplers reduced the glucose molar growth yields (Ygluc) of B. ruminicola, B. succinogenes, and B. fibrisolvens compared with those obtained without uncouplers. The extent of Ygluc reduction accompanying inhibitor exposure reflected electron transport chain structure. S. ruminantium appeared to obtain its adenosine 5'-triphosphate from substrate-level processes only. The other organisms studied appeared to obtain adenosine 5'-triphosphate both from substrate-level processes and from electron transport but differed in the amount of adenosine 5'-triphosphate obtained from glucose catabolism and in the proportions of adenosine 5'-triphosphate obtained from substrate-level reactions and electron transport.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system.

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Utilization of xylooligosaccharides by selected ruminal bacteria.

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Inhibitory effects of H2 on growth of Clostridium cellobioparum.

Hydrogen inhibits the growth of hydrogen-producing Clostridium cellobioparum, but not of Escherichia coli or Bacteroides ruminicola. The inhibition is reversible. When hydrogen was removed either by palladium black or by gassing out the tube, glucose utilization increased as did optical density and hydrogen production of C. cellobioparum. Removal of the H2 by methanogenic bacteria favors the growth of C. cellobioparum. Grown with Methanobacterium ruminantium in various concentrations of glucose, the Clostridium reaches a higher optical density and produces more H2 and a higher viable cell count. The cell yield is also higher than in pure culture. In mixed culture, C. cellobioparum produces more acetic acid and less lactic acid, ethanol, and butyric acid than in pure culture. The significance of this metabolic shift and hydrogen utilization in methanogenesis is discussed.     

Activity and properties of fumarate reductase in ruminal bacteria

Fumarate-reducing bacteria were sought from the main ruminal bacteria. Fibrobacter succinogenes, Selenomonas ruminantium subsp. ruminantium, Selenomonas ruminantium subsp. lactilytica, and Veillonella parvula reduced fumarate by using H(2) as an electron donor. Ruminococcus albus, Prevotella ruminicola, and Anaerovibrio lipolytica consumed fumarate, although they did not oxidize H(2). Of these bacteria, V. parvula, two strains of Selenomonas, and F. succinogenes had a high capacity to reduce fumarate. In all the fumarate-reducing bacteria examined, fumarate reductase existed in the membrane fraction. Based on the activity per cell mass and the affinity of fumarate reductase to fumarate, these bacteria were divided into two groups, which corresponded to the capacity to use H(2): A group of bacteria with higher activity and affinity were able to use H(2) as an electron donor for fumarate reduction. The bacteria in this group should gain an advantage over the bacteria in another group in fumarate reduction in the rumen. Cellulose digestion by R. albus was improved by fumarate reduction by S. lactilytica as a result of an increased growth of R. albus, which may have been caused by the fact that S. lactilytica immediately consumed H(2) produced by R. albus. Thus fumarate reduction may play an important role in keeping a low partial pressure of H(2) in the rumen.     

Effects of the Fusarium spp. mycotoxins fusaric acid and deoxynivalenol on the growth of Ruminococcus albus and Methanobrevibacter ruminantium

The Fusarium spp. mycotoxins fusaric acid and deoxynivalenol (DON) were tested for antimicrobial activity against Ruminococcus albus and Methanobrevibacter ruminantium. The growth of both organisms was inhibited by fusaric acid as low as 15 micrograms/mL (84 microM) but not by DON, at levels as high as 100 micrograms/mL (338 microM). No synergistic inhibitory effect was observed with DON plus fusaric acid. Neither organism was able to adapt to the fusaric acid and responses of each organism to the compound were different. The optical density (OD) maximum for R. albus, but not for M. ruminantium, was diminished after 28 days incubation at concentrations of fusaric acid below 240 micrograms/mL. Inhibition of R. albus started before significant growth had occurred, while M. ruminantium doubled twice before the onset of inhibition. Responses to picolinic acid, an analog of fusaric acid, were also dramatically different between the two microorganisms with M. ruminantium exhibiting a severe lag followed by a complete recovery of growth, while R. albus was only slightly inhibited with no lag. These results suggest that the mechanism of fusaric acid inhibition is specific to each microorganism. This is the first demonstration of the common mycotoxin fusaric acid inhibiting the growth of rumen bacteria.     

Effects of Combinations of Substrates on Maximum Growth Rates of Several Rumen Bacteria

Five rumen bacteria, Selenomonas ruminantium, Bacteroides ruminicola, Megasphaera elsdenii, Butyrivibrio fibrisolvens, and Streptococcus bovis were grown in media containing nonlimiting concentrations of glucose, sucrose, maltose, cellobiose, xylose and/or lactate. Each bacterium was grown with every substrate that it could ferment in every possible two-way combination. Only once did a combination of substrates result in a higher maximum growth rate than that observed with either substrate alone. Such stimulations of growth rate would be expected if specific factors unique to individual substrates (transport proteins and/or enzymes) were limiting. Since such synergisms were rare, it was concluded that more general factors limit maximum growth rates in these five bacteria.     

Effect of mercuric ion on the growth, photosynthesis, and nitrogenase activity of Anabaena inaequalis.

Maintenance energy expenditures were mesured for five rumen bacteria, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Bacteroides ruminicola, Megasphaera elsdenii, and Streptococcus bovis, by using a complex medium with glucose as the carbon source. Large differences (as high as 8.5-fold) in maintenance energy expenditures were seen among these bacteria. The suggestion is made that maintenance requirements could be a significant determinant of bacterial competition in the rumen. Theoretical maximum growth yields, calculated from double reciprocal plots of yield versus dilution rate, were compared to theoretical Y_ATP^max values in order to estimate minimum molar adenosine 5′-triphosphate yields from glucose for each bacterium. Results showed that relative yield among the bacteria was growth rate dependent. At high dilution rates, both S. ruminantium and S. bovis produced lactate as their principal fermentation product. At lower dilution rates very little lactate was formed and growth yields increased. Acetate and ethanol were the predominant fermentation products of S. bovis at low dilution rates. Other workers have shown that S. ruminantium produces acetate and propionate at low growth rates.     

Effects of methanol on the growth of gastrointestinal anaerobes

The effects of methanol on the growth of representative, predominant, anaerobic gut bacteria were studied. Growth yields and rates were determined in a base medium to which methanol was added to produce media with methanol concentrations varying, in twofold steps, over a concentration range of 0.01 to 25%, by volume. The growth of many of the organisms was completely inhibited by a methanol concentration equal to, or less than, 6.2%. Isolates representing cellulolytic species were completely inhibited at a methanol concentration of 3.1%, and inhibitory effects on the yield of some cellulolytic isolates were found at a methanol concentration as small as 0.01%. Although most of the organisms studied were inhibited at relatively small methanol concentrations, isolates of Selenomonas ruminantium, Bacteroides ovatus, and Fusobacterium necrophorum were relatively methanol resistant. A methanol concentration of 12.5% was required to completely inhibit S. ruminantium. Substantial growth of B. ovatus was obtained in media containing 12.5% methanol, and for F. necrophorum, substantial growth occurred in media containing 25% methanol. The yields of F. necrophorum strain B85 and S. ruminantium strain PC18 were enhanced by relatively small methanol concentrations and reduced with further methanol concentration increase Anaerobic, nonsporing gut bacteria exhibit a diversity of responses to methanol.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

GLUCOSE FERMENTATION AND GROWTH OF SELENOMONAS SPUTIGENA ON A MINIMAL MEDIUM

Very little is known about the growth physiology and metabolic niche of the human oral isolate Selenomonas sputigena. The objective of this study was to devise a minimal medium for comparing growth rates and fermentation of rumen Selenomonas ruminantium strains with S. sputigena. When anaerobically grown on a minimal glucose medium containing yeast extract as the only chemically undefined component, S. sputigena produced acetate, propionate, and succinate while S. ruminantium strains produced primarily lactate. When strains were compared (P < 0.05) for each carbon source that yielded growth, rumen strain HD₄ grew faster than all other strains on glucose, cellobiose and glycerol while strain GA192 grew faster on trehalose. Rumen strains GA192, PC18, and HD₄ grew faster on mannitol than rumen strains D and GA31. S. sputigena grew faster on lactate (0.38 ± 0.04) than any of the S. ruminantium strains. The minimal medium developed in this study should be useful for jurmer physiological studies on fermentation and metabolism in S. sputigena.     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.