Efficiency of energy utilization by mixed rumen bacteria in continuous culture.

Mixed bacterial cultures derived from the rumen were grown in a remen fluid medium in a chemostat at three dilution rates (.02, .06, and .12 per h), each at four growth-limiting glucose concentrations (5.8, 9.9, 12.7, and 25.0 mM). Microscopic observations indicated that a relatively complex mixture of bacterial species was maintained and proportions of fermentations products were similar to those of the rumen except for elevated proportions of methane and acetate. Cell concentration increased linearly with increases in glucose concentration. The range of glucose concentrations had little effect on yields of cells or products produced per mole of glucose fermented. With increases in dilution rates, the amount of butyrate and methane produced per mole of glucose fermented decreased and the amount of propionate increased. Yield glucose (grams cells produced per mole of glucose fermented) increased from 42 at a dilution rate of .02 to 84 at a dilution rate of .12. These large increases are discussed in relationship to the energy requirements for maintenance of bacteria. A theoretical maximum yield glucose of 89.3 and a maintenance requirement of .26 mmol glucose per g cells per h were calculated. Moles of adenosine triphosphate produced per mole of glucose fermented and yield of cells produced per mole of adenosine triphosphate are discussed.     

Concentration of ammonia across cell membranes of mixed rumen bacteria

When mixed ruminal bacteria were provided with growth rate limiting amounts of mixed carbohydrates, more than 50 mg ammonia/L were required for maximal protein synthesis. Microbial protein synthesis declined when ammonia concentration was less than 50 mg/L and unfermented carbohydrates increased. Ammonia starvation also decreased growth efficiency. Intracellular ammonia increased as a linear function of extracellular ammonia, but the intracellular concentration was always at least 160 mg/L higher than the extracellular concentration. Maximal protein synthesis was not observed until intracellular ammonia was greater than 220 mg/L. The concentration gradient of ammonia across cell membranes ranged from 15-fold to 1.8-fold and indicated that some of the ruminal bacteria may have active transport mechanisms for ammonia. These concentration gradients were, however, far less than those reported for bacteria from other habitats. The ruminal bacteria left more than 12 mg ammonia/L when carbohydrates were still available, and this observation was consistent with the assumption that active ammonium transport was not readily or maximally induced.     

Effect of carbon-4 and carbon-5 volatile fatty acids on growth of mixed rumen bacteria in vitro

Mixed ruminal bacteria (400 mg cells/liter) were incubated in artificial media containing ammonia, sodium carbonate, macrominerals, vitamins, sulfide, microminerals, acetate, propionate, and butyrate. When mixed carbohydrates (equal parts glucose, maltose, sucrose, cellobiose, and soluble starch) were added at 155 mg/liter per h for 10 h, average bacterial growth rate was slow, and dry weight yield was greater than 23%. Additions of isobutyrate, valerate, isovalerate, and 2 methylbutyrate had little influence on synthesis of bacterial dry weight, deoxyribonucleic acid, ribonucleic acid, or carbohydrate. When a timothy hay inoculum was used, isovalerate and 2 methyl-butyrate increased protein synthesis by 11.2 and 16.4%, but isobutyrate and valerate alone were without effect. All four acids combined increased bacterial protein by 18.7%. Responses with an inoculum of 60% concentrate and mixed hay were smaller and not statistically different from control incubations. Low concentrations of Trypticase (less than 250 mg/liter) improved efficiency of microbial protein synthesis from organic matter, but more was associated with decreased efficiency and utilization of extracellular ammonia.     

Effect of inoculum preparation and dietary energy on microbial numbers and rumen protein degradation activity

Chilling of whole rumen contents prior to preparation of strained rumen fluid enriched with particle-associated microorganisms resulted in rumen inoculum with the highest microbial dry weight, total bacterial counts, and degradation rates for casein and soybean meal. Strained rumen fluid, whole rumen contents blended with strained rumen fluid, chilled strained rumen fluid, and strained rumen fluid plus particle-associated microorganisms were lower in bacterial counts and microbial dry weight. Except for strained rumen fluid plus particle-associated microorganisms, protein degradation rates were also lower. Three ruminally cannulated cows were used in a 3 X 3 Latin square experiment to determine the effect of diet on rumen microbial numbers and protein degradation rates. Cows were fed the following diets ad libitum: 1) 100% alfalfa hay (20.7% crude protein); 2) 63% alfalfa hay and 37% corn-soybean meal concentrate (18.2% crude protein); and 3) 37% alfalfa hay and 63% corn-soybean meal concentrate (15.1% crude protein). Diet 3 yielded rumen contents with the highest concentration of microbial dry weight, total and viable bacterial counts, total protozoal counts, and fractional degradation rates for casein and bovine serum albumin. However, degradation rate per unit microbial dry matter was not altered by diet.     

Alteration of fermentation in continuous culture of mixed rumen bacteria by isolated alfalfa saponins

Saponins isolated from alfalfa by ethanol extraction and acid hydrolysis were incorporated into protein-free purified diets at 0, .5, 1, 2, and 4% of dietary dry matter to investigate the effect of saponins on fermentation by mixed rumen bacteria maintained in continuous culture. Inoculum was obtained from a lactating dairy cow fed alfalfa hay and a 13% crude protein grain mixture with forage to grain ratios of 33:67, 67:33, or 100:0. Outflow of microbial protein was lower in fermenters fed purified diet containing 1% saponins. Accumulation of ammonia was observed in fermenters fed purified diets containing alfalfa saponins. Total volatile fatty acid production was reduced by addition of isolated alfalfa saponin fraction at .5, 1, 2, and 4% concentrations. Acetate to propionate ratios were reduced from 1.93 in control to 1.37 in fermenters with 1% saponins. Extent of change in fermentation was not proportional to the concentration of added saponins.     

The effects of urea and artificial saliva on rumen bacterial protein synthesis in sheep receiving a high-cereal diet

Four sheep fitted with cannulas in the rumen and duodenum and given a basal diet of 18% chopped hay, 41% rolled barley and 41% flaked maize, known to be associated with low rates of bacterial protein synthesis in the rumen, were used in a 4 × 4 Latin Square experiment to study the effects of continuous intraruminal infusions of water (2 litres day^?1; control), urea solution (7.5 g urea litre^?1, 2 litres day^?1; urea), artificial saliva (4 litres day^?1; saliva) and artificial saliva with added urea (3.75 g urea litre^?1, 4 litres day^?1; saliva+urea) on rumen ammonia-nitrogen concentration, pH, liquid clearance rates and bacterial protein synthesis. Rumen ammonia-nitrogen concentrations for control, urea, saliva and saliva + urea treatments were 81, 158, 38 and 151 mg litre^?1, respectively. Corresponding mean values for rumen pH were 6.18, 6.09, 6.37 and 6.41 units and for rumen liquid clearance rates 0.082, 0.057, 0.062 and 0.051 h^?1. The mean rate of bacterial protein synthesis in the rumen, estimated from the duodenal entry of α-?-diaminopimelic acid, for the control treatment was 163 g crude protein (CP) kg^?1 organic matter (OM) apparently digested in the stomach. Corresponding rates for the saliva and saliva + urea treatments were 169 g CP kg^?1 OM apparently digested and 215 g CP kg^?1 OM apparently digested, indicating responses in protein synthesis to the urea+saliva treatment but not to the saliva alone treatment. Infusion of urea solution increased the mean rate of protein synthesis to 204 g CP kg^?1 OM apparently digested in the stomach but the responses were variable between animals and in three of the animals were small. It is concluded that with the type of diet used an enhanced entry of saliva into the rumen is necessary to ensure that supplementary urea produces a consistent improvement in ruminal protein synthesis.     

Enrichment and characterization of chlorinated organophosphate ester-degrading mixed bacterial cultures

Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.     

Ammonia concentration and protein synthesis in the rumen

The effect of rumen ammonia concentration on microbial protein synthesis and fermentation was studied in three separate experiments. In each experiment three separate sheep were fed semi-purified diets [designated A (‘concentrate’), B (‘roughageconcentrate’) or C (‘roughage’)] and infused intra-ruminally with five graded amounts of urea according to a randomised block design. Rumen ammonia concentrations remained low until the total nitrogen intake was about 10 g day^?1 after which rumen ammonia concentration rose rapidly. Rumen and duodenal ammonia concentrations were linearly related (r=0.90, 0.98 and 0.84 for diets A, B and C, respectively; P< 0.001). Microbial protein production did not increase when rumen ammonia concentrations exceeded 2.8 mM for diet A, 6.0 mM for diet B and 1.6 mM for diet C. Diets A and C produced a propionate-type fermentation while diet B was characterised by an acetate-type fermentation. Rumen ammonia concentration had no apparent effect on either concentration or the molar proportions of volatile fatty acids. There were no systematic trends in digestibility in relation to rumen ammonia concentration.     

pH dynamics and bacterial community composition in the rumen of lactating dairy cows

The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6 h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH = 6.11 to 6.51, diurnal pH range = 0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean =  <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii.     

Metabolites produced during fermentation of wine by mixed cultures of yeasts and lactic acid bacteria

Certain aspects of the interrelationship between associations of yeasts and lactic acid bacteria during the fermentation of grape must were studied. Action bySaccharomyces pastorianus gave rise to increased formation of keto acids and residual metabolites. Addition ofLactobacillus plantarum to the fermentation process may inhibit alcoholic fermentation and considerably increased the production of acetaldehyde andl(+) andd(–)lactic acids.

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Entstehung von Metaboliten bei der Fermentation von Wein durch Mischkulturen von Hefen und Milchsäurebakterien

Zusammenfassung Es wurden Wechselbeziehungen zwischen Hefen und Milchsäurebakterien während der Fermentation von Traubensaft untersucht.Saccharomyces pastorianus bewirkte ein Ansteigen der Ketosäurenproduktion und der Bildung von Rest-Metaboliten. Das Hinzufügen vonLactobacillus plantarum beim Gärungsprozess kann die alkoholische Gärung unterdrücken und hat die Produktion von Acetaldehyd sowie vonl(+)- undd(–)-Milchsäuren beträchtlich gesteigert.

     

Metabolism of peptides and amino acids during in vitro protein degradation by mixed rumen organisms

In vitro inoculum enriched with particle-associated organisms was prepared using rumen contents from a cow fed a 60% forage, 40% concentrate diet. Treatment of in vitro inoculum with cetyltrimethylammonium bromide was used to release intracellular free amino acids from mixed rumen organisms. Addition of 10 mM tosylarginine methyl ester, a competitive inhibitor of trypsin, decreased degradation rate and intracellular free amino acids in incubations containing either casein or serum albumin. Extracellular peptides increased rapidly to a maximum at 60 min in casein incubations but were not different from zero in albumin incubations. Accumulation of intracellular free amino acids was maximal at 60 min in casein and albumin incubations; the concentration observed with albumin was about one-fourth that with casein. Ammonia production from intact casein was slightly greater than that from acid and enzymatically hydrolyzed casein and about 80% greater than that from albumin. Ammonia production and appearance of extracellular free amino acids lagged behind accumulation of intracellular free amino acids. Results suggest that formation and metabolism of extracellular peptides are important in controlling the rate of protein degradation by mixed rumen organisms.     

Effect of diet on amino and nucleic acids of rumen bacteria and protozoa

Amino acid composition and nucleic acid content of pure cultures of rumen bacteria (17 species) were analyzed. Amino acid composition between gram-positive and -negative organisms was not different. The total nitrogen content of gram-negative bacteria (10.8%) was significantly higher than gram-positive organisms (9.9%). Deoxyribonucleic acid-nitrogen: total nitrogen (mg/g) differed between gram-positive (8.8) and gram-negative (18.9) bacteria, but there was no significant difference in ratio of ribonucleic acid-nitrogen to total nitrogen. In a second experiment six rumen-fistulated cattle were fed either a high roughage (85% alfalfa hay plus 15% concentrate) or high concentrate diet (15% alfalfa hay and 85% concentrate). Cattle were adapted 14 days and rumen contents sampled on 3 consecutive days. Nitrogen content was higher in protozoa from cattle fed low concentrate (8.4%) than in protozoa from cattle fed high concentrate (7.9%) but was similar in bacteria for both diets. Deoxyribonucleic acid nitrogen: total nitrogen (mg/g in bacteria decreased from 27.2 in cattle fed the low concentrate diet to 20.9 in those fed the high concentrate diet. Differences between sampling days were significant for both bacteria and protozoa for ratio of deoxyribonucleic acid-nitrogen to total nitrogen but were significant only in protozoa for ratio of ribonucleic acid nitrogen to total nitrogen. Ribonucleic acid may serve as a marker for estimating microbial production in the rumen if sources of variation are recognized and corrected adequately.     

Studies on rumen metabolism. V.—formation of branched long chain fatty acids in cultures of rumen bacteria

When mixed rumen bacteria were incubated with isobutyrate-1-¹⁴C, the ¹⁴C-label was incorporated into branched long chain fatty acids with both odd and even numbers of carbon atoms. Tracer studies showed that the ¹⁴C-label from DL-valine-4-¹⁴C was incorporated into branched long chain fatty acids and also into straight chain fatty acids with odd numbers of carbon atoms. In addition, labelled isobutyrate and propionate were synthesised from valine and probably represent intermediates in the synthesis of the branched and straight chain fatty acids respectively.     

Efficiency of bacterial protein synthesis in the rumen of sheep receiving a diet of sugar beet pulp and barley.

Eight sheep fitted with both rumen and duodenal re-entrant cannulae were used to investigate the digestion of a diet consisting of 60% molassed sugar beet pulp and 40% ground barley. Molar proportions of acetic acid in the rumen varied from 55 to 66%, of propionic acid from 15 to 26% and of butyric acid from 12 to 19%. On average, 69.1 ± 1.2% of the dietary organic matter was digested in the rumen and 89.9 ± 0.4% in the whole digestive tract. Corresponding figures for gross energy were 64.4 ± 1.4% and 87.4 ± 0.4%, for cellulose 82.8 ± 1.4% and 86.3 ± 0.8% and for α-linked glucose polymers were 91.6 ± 0.9% and 100 ± 0%. There were only small differences in the extent of digestion of these constituents in the rumen between animals and the mean production of total short-chain fatty acids was 55.5 ± 1.6% of the digestible energy. The mean daily amount of nitrogen entering the duodenum was 11.1 ± 5.1% greater than the dietary intake and the apparent digestibility of nitrogen was 76.7 ± 1.0%. Calculation of bacterial protein using α-±-diaminopimelic acid as a marker, indicated that bacterial protein constituted about 80% of the total protein entering the duodenum and that the average efficiency of rumen protein synthesis was 14.31 ± 0.75 g crude protein/100 g OM “disappearing” in the rumen. With the exception of one animal in which the rumen ammonia concentration was especially high, the efficiency of protein synthesis varied between animals over only a narrow range and there was no evidence that efficiency was influenced by variation in the pattern of fermentation in the rumen.     

不同pH值培养乳酸球菌对其发酵活力的影响

为了确定嗜热链球菌St-1最适生长pH值,研究了在不同pH值条件下培养嗜热链球菌St-1,对其活菌数、发酵活力及菌体形态学特征的影响.结果表明,当培养体系的pH值恒定在6.0时,其活菌数和发酵活力最高;对比发酵活力曲线和生长曲线表明,发酵活力最好时的活菌数并不是最高的;对比不同恒定pH值条件下培养活力最高时的嗜热链球菌St-1菌体形态特征发现,嗜热链球菌St-1的链越长,其发酵活力越低,链越短发酵活力越高.     

Phagocytic and postphagocytic activities of bovine neutrophils for pure and mixed bacterial cultures

A comparative study of phagocytosis and postphagocytic oxidative metabolic activity of bovine blood neutrophils incubated with pure and mixed cultures of Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae was preformed. Most neutrophils when incubated with mixed cultures showed preferential phagocytosis for one species and a smaller number phagocytized both species of microorganisms. Percent phagocytosis for E. coli in pure culture was similar to that of Strep. agalactiae in pure culture and higher than that for Staph. aureus in pure culture. Neutrophils incubated with mixed cultures of E. coli and Staph. aureus or E. coli and Strep. agalactiae showed greater than expected phagocytosis of each microorganisms alone and reduced phagocytosis of both microorganisms together. Postphagocytic oxidative metabolic activity of neutrophils, measured by percent nitroblue tetrazolium reduction, did not differ following phagocytosis of these three microorganisms in pure cultures. In comparison, a synergistic effect on nitroblue tetrazolium reductive activity was seen in mixed cultures as evidenced by higher percent nitroblue tetrazolium reduction following phagocytosis of both E. coli and Staph. aureus or E. coli and Strep. agalactiae. These observations indicate that the phagocytic and metabolic activities of neutrophils for bacteria in mixed cultures may not be identical to those in pure cultures.     

Studies on rumen metabolism. 8. Characteristics of lipases in rumen contents and in rumen bacteria

Soluble lipases obtained from the rumen contents of pasture-fed cows had pH optima of 7.0 and 8.0 to 8.5, respectively, with tributyrin and triolein as substrates. At these optima the activity towards tributyrin was about 30 times greater than towards triolein. Tributyrin-splitting bacteria were abundant in the rumen of pasture-fed cows but none of the three strains isolated were able to hydrolyse triolein. One of these strains showed a limited capacity to de-esterify galactolipid.     

Rheology of whey protein isolate-xanthan mixed solutions and gels. Effect of pH and xanthan concentration

Flow properties at pH 5.5-7.5 of whey protein isolate (WPI)-xanthan solutions containing 0-0.5 w/w% xanthan were studied by viscosimetry, although rigidity and fracture properties of the corresponding heat-set gels (90°C, 30 min) were determined by uniaxial compression. All the studied solutions displayed generalized shearthinning flow behaviour. Synergistic WPI-xanthan interactions has been revealed by observing that rheological parameters [σ_msf, K, n, η (γ)] characterizing blends were larger than those calculated from the two separated solutions. Such a behaviour was attributed to segregative phase separation of whey proteins and xanthan. Effects of xanthan on WPI-xanthan gel properties both depended on pH and xanthan concentration. Simultaneous increased xanthan concentration and decreased pH inhibited gelation of WPI-xanthan blends. Regarding gel strength, synergistic WPI-xanthan interactions were observed at pH >7.0 and low xanthan concentration (0.05 or 0.1 w/w%). Antagonism between the two macromolecules occurred at low xanthan concentration and pH ≤6.5, and high xanthan concentration (0.2 or 0.5 w/w%) at all pH tested. Low xanthan concentration rendered mixed gels more brittle than protein gels, and high xanthan concentration decreased pH effects on gel stress-strain relationships. The balance between strong thermal aggregation of concentrated whey proteins - in presence of incompatible xanthan -, high viscosity of blends and repulsive surface forces of protein molecules was thought to be at the origin of WPI-xanthan gel mechanical properties.