Relationship of lactate dehydrogenase specificity and growth rate to lactate metabolism by Selenomonas ruminantium.

A lactate-fermenting strain of Selenomonas ruminantium (HD4) and a lactatenonfermenting strain (GA192) were examined with respect to the stereoisomers of lactate formed during glucose fermentation, the stereoisomers of lactate fermented by HD4, and the characteristics of the lactate dehydrogenases of the strains. GA192 formed L-lactate and HD4 formed L-lactate and small amounts of D-lactate from glucose. HD4 fermended L- but not D-lactate. Both strains contain nicotinamide adenine dinucleotide (NAD)-specific lactate dehydrogenases, and no NAD-independent lactate oxidation was detected. Continuous cultures of both strains grown with limiting glucose produced mainly propionate and acetate and little lactate at dilution rates less than 0.4/h, with shifts to increasing amounts of lactate and less acetate and propionate as the dilution rate was increased from 0.4/h to approximately 1/h.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

Changes in metabolism of the rumen bacterium Streptococcus bovis H13/1 resulting from alteration in dilution rate and glucose supply per unit time

Streptococcus bovis H13/1 was grown in a glucose-limited chemostat. A concomitant increase in dilution rate and glucose supply per unit time caused both an increase in lactate production per mole of glucose fermented and a linear increase in growth yield over the dilution rate range 0.052 to 0.141/h. When the dilution rate was increased with no change in glucose supply per unit time there was a reduction in lactate production and an increase in that of acetate and ethanol coinciding with a non-linear increase in growth yield. YMaxglu = 38.6 and a maintenance coefficient, ms = 0.290 mmol/l glucose/g cells/h were calculated. The results also suggested an interaction between the formate and CO2 pools.     

Changes in metabolism of the rumen bacterium Streptococcus bovis H13/1 resulting from alteration in dilution rate and glucose supply per unit time

Streptococcus bovis H13/1 was grown in a glucose-limited chemostat. A concomitant increase in dilution rate and glucose supply per unit time caused both an increase in lactate production per mole of glucose fermented and a linear increase in growth yield over the dilution rate range 0.052 to 0.141/h. When the dilution rate was increased with no change in glucose supply per unit time there was a reduction in lactate production and an increase in that of acetate and ethanol coinciding with a non-linear increase in growth yield. Y_glu^Max= 38.6 and a maintenance coefficient, m_s= 0.290 mmol/l glucose/g cells/h were calculated. The results also suggested an interaction between the formate and CO₂ pools.     

The kinetics of glucose fermentation bySelenomonas ruminantium HD4 grown in continuous culture

Summary The production of organic acids (acetate, lactate, and propionate) by the anaerobic, ruminal bacteriumSelenomonas ruminantium HD4 was investigated in both glucose-limited and glucose-sufficient (phosphate-limited) continuous cultures. The fermentation pattern of products exhibited a shift upon release of glucose limitation from acetate and propionate to lactate at a dilution rate of 0.2 h^–1. Glucose sufficiency brought about two-to fourfold increase in specific glucose utilization rate, lactate productivity, and lactate yield relative to glucose-limited growth conditions. The increased lactate production under glucose-sufficient growth conditions was attributed to the overutilization of excess glucose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

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.     

Production of acetic acid by Acetogenium kivui

Summary The formation of acetic acid by the thermophilic nonsporeforming homoacetogenic bacterium Acetogenium kivui was studied under various conditions. In pH-controlled batch fermentation at pH 6.4 this bacterium was able to produce up to 625 mM of acetic acid from glucose within 50–60 h. The value of _max obtained was about 0.17 h^-1, the yield was about 2.55 mol of acetic acid per mol of glucose utilized. In continuous fermentation both substrate concentration and dilution rate (D) influenced the yield of acetate and the stationary concentration: a glucose concentration of 67 mM at D=0.09 h^-1 resulted in 2.82 mol acetate/mol glucose and 190 mM acetate at a production rate of 17.1 mM/1 h. When the dilution rate was increased the production rate reached a maximal value of 43.2 mM/1 h at D=0.32 h^-1. At a glucose concentration of 195 mM the dependence of yield upon dilution rate followed a similar pattern and an acetate concentration of 420 mM could be obtained. Enzymatic studies indicate that in A. kivui pyruvate ferredoxin-oxidoreductase and acetate kinase are inhibited at acetate concentrations higher than 800 mM. Based on these results a fed-batch fermentation was developed, which allowed to produce more than 700 mM acetic acid within 40–50 h.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach

The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, Q _O2, remained between 14–18 mmol O₂ h^-1 g dwt^-1 and the maximum growth rate varied from 0.22 h-¹ on acetate to 0.77 h^-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µ_crit, the growth yield Y_XO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of m_ATP < 4 mmol ATP h^-1 g dwt^-1 determined from A-stat cultivation data was low compared with the calculated unproductive hydrolysis of ATP (64–103 mmole ATP g dwt^-1).     

Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures

Saccharomyces uvarum, Geotrichum candidum, Endomycopsis burtonii and Hansemula canadensis have been isolated from deteriorated silages and/or spoiled lactic acid fermented foods. All yeasts could grow in relatively high (100 mmol/l) concentrations of acetate or lactate in Wickerham's assimilation broth but were inhibited by increasing concentrations of these acids. Propionate was much more inhibitory than acetate or lactate. Growth rate, but not final cell yield, was adversely affected by increasing concentrations of these acids. The three acids used in combination were synergistically inhibitory to H. canadensis and G. candidum. Saccharomyces uvarum was inhibited by synergistic mixtures of acetate/propionate. Endomycopsis burtonii and H. canadensis were inhibited by synergistic mixtures of acetate/lactate, acetate/propionate and lactate/propionate. Lactate and propionate had a synergistic inhibitory effect on G. candidum. The mechanism of inhibition of growth of S. uvarum by acetate and propionate was further investigated. Acetate and propionate each caused a reduction in cellular efficiency (Y, μg cells/μmole glucose consumed). Mixtures caused a drastic reduction in growth rate and Y. These results suggest that the cells may expend energy in reducing intracellular concentrations of acetate while propionate probably acts in a different manner.     

Cell yields and fermentation responses of a <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium poultry isolate at different dilution rates in an anaerobic steady state continuous culture

The objectives of these studies were to determine cell yield and fermentation responses of a Salmonella enterica serovar Typhimurium poultry isolate using various dilution rates in steady state continuous culture incubations. S. enterica Typhimurium cells were propagated in continuous cultures with a total volume of 0.50 l of Luria Bertani medium containing 0.1% glucose. Dilution rates from 0.0125 to 1.44/h were used. Cell protein concentration generally increased linearly with increased dilution rate up to a rate of 0.54/h and declined at the higher dilution rates. Glucose consumption gave a similar pattern to cell protein concentration by declining at the three highest dilution rates. Short chain fatty acid production was inconsistently influenced by dilution rate. Acetate, the most predominant fatty acid produced, declined at the higher dilution rates, as did propionate. Ammonia production remained stable at the lowest dilution rates, but increased significantly at a dilution rates above 0.27/h.     

Combined effect of salinomycin and feeding on whole body glucose kinetics in sheep fed a high-concentrate diet

The aim of this study was to investigate the effects of salinomycin (SL) and feeding on whole body glucose kinetics in sheep fed a high-concentrate diet (25% orchardgrass hay and 75% commercial concentrate). Four adult sheep were fed the diet with or without 20 mg x kg(-1) diet of SL once daily for each 3 wk. The rates of glucose entry and utilization were determined before and during 3 h after feeding using a [ (13)C(6)] glucose dilution approach. Ruminal characteristics and concentrations of blood volatile fatty acids (VFA) and plasma glucose and insulin were also measured. Metabolizable energy intake was unaffected (P = 0.22) with SL. Salinomycin decreased (P = 0.06) the ratio of acetate to propionate in rumen fluid. Salinomycin increased (P = 0.01) both rates of entry and utilization of glucose, but did not affect (P > 0.10) concentrations of blood VFA or plasma glucose or insulin. Feeding caused gradual increases in concentrations of blood acetate (P < 0.01) and propionate (P = 0.01), a transient increase in plasma insulin concentration (P = 0.05), a transient decrease in plasma glucose concentration (P < 0.01), and persistent increases in both rates of glucose entry (P < 0.01) and utilization (P < 0.01). No SL x feeding interaction was observed (P > 0.10) on any measurements. We conclude that SL and feeding would have an additive effect on both rates of glucose entry and utilization without modifications with SL to feeding responses of peripheral concentrations of blood VFA, plasma glucose and insulin.     

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.     

Growth and fermentation responses of Selenomonas ruminantium to limiting and non-limiting concentrations of ammonium chloride

 The objective of this study was to assess fermentation product, growth rate and growth yield responses of Selenomonas ruminantium HD₄ to limiting and non-limiting ammonia concentrations. The ammonia half-inhibition constant for S. ruminantium in batch culture was 296 mM. Cells were grown in continuous culture with a defined ascorbate-reduced basal medium containing either 0.5, 5, 25, 50, 100 or 200 mM NH₄Cl and dilution rates were 0.07, 0.14, 0.24 or 0.40 h^-1. Ammonia was the growth-limiting nutrient when 0.5 mM NH₄Cl was provided and the half-saturation constant was 72 μM. Specific rates of glucose utilization and fermentation acid carbon formation were highest for 0.5 mM NH₄Cl. Lactate production (moles per mole of glucose disappearing) increased at the fastest dilution rate (0.40 h^-1) for 5.0 mM NH₄Cl while acetate and propionate decreased when compared to slower dilutions (0.07 and 0.14 h^-1). Lactate production remained low while acetate and propionate remained high for all dilution rates when NH₄Cl concentrations were 25 mM or greater. Yield (Y _Glc and Y _ATP) were nearly doubled when NH₄Cl was increased from 0.5 mM (25.1 g cells/mol glucose used and 13.9 g cells/mol ATP produced respectively) to the higher concentrations. Y _Glc was highest at 25 mM and 50 mM NH₄Cl (48.2 cells/mol and 43.1 cells/mol respectively) as was Y _ATP (23.2 cells/mol and 20.8 cells/mol respectively). Y _NH3 was highest at the lowest NH₄Cl concentration. The maximal fermentation product formation rate occurred at a growth-limiting ammonia concentration, while maximal glucose and ATP bacterial yields occurred at non-growth-limiting ammonia concentrations. Given the growth response of this ruminal bacterium, it is possible that maximization of ruminal bacterial yield may necessitate sacrificing the substrate degradation rate and vice versa. Received: 5 December 1995/Received revision: 2 April 1996/Accepted: 22 April 1996     

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.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80. The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM. At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed. The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S. cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition. The major difference between the two yeast strains was that S. cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid. The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase. The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed. This resulted in a reduction in yield and an increase in specific ethanol production. Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.     

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.     

The co-disposal of a model brewery wastewater with domestic refuse

By use of refuse columns, it was shown that, at appropriate organic loading rates, the co-disposal of a synthetic brewery wastewater with refuse stimulated methane production and did not reduce leachate quality, in terms of pH or volatile fatty acid concentrations. When the application rate was doubled, from imposed dilution rate ( D ) 0·025 to 0·056 h⁻¹, there was no breakthrough of volatile fatty acids and methane production was promoted. However, when the same total organic load was achieved by doubling the organic strength of the wastewater, but maintaining D = 0·025 h⁻¹, leachate quality was temporarily reduced, with elevated concentrations of acetate and propionate.     

Hydrolysis and acidogenic fermentation of a protein,gelatin, in an anaerobic continuous culture

Summary Model studies of anaerobic protein digestion were performed using gelatin dissolved in a mineral medium, which was fed to a mixed population of bacteria in a carbon-substrate limited chemostat culture. The dilution rate and culture pH value were varied progressively in order to determine the optimal conditions for hydrolysis and acidification (i.e., fatty acids formation). The optimum pH value appeared to be in the neutral region (pH>6.3), and the maximal dilution rate allowing steady state growth was 0.23 h^-1. At this dilution rate and at pH 7 hydrolysis of gelatin was 78% complete, and 79% of the protein hydrolysed was fermented to identifiable products. At submaximal dilution rates both these values were higher. The main fermentation products were acetate, propionate, and valerate, and minor amounts of other volatile fatty acids. The product composition was relatively independent of the dilution rate, but varied substantially with the pH value.