Influence of glucose on glycerol metabolism by wild-type and mutant strains of Clostridium butyricum E5 grown in chemostat culture

In order to improve the yield of 1,3-propanediol (1,3-PPD) in Clostridium butyricum E5, we carried out cofermentation experiments on glucose/glycerol mixtures in chemostat culture. The results showed the influence of the ratio of the two carbon substrates on the production of the required diol. The progressive increase of glucose in culture medium containing a given concentration of glycerol made it possible to highlight the deviation of carbon flow from the oxidative towards the reducing pathway, in order to maintain the oxidation/reduction balance in the cell. The conversion of glycerol into 1,3-PPD thus increased from 0.63 mol mol(-1), without the addition of glucose, to a maximum of 0.89 mol mol(-1) for a molar glucose/glycerol ratio of 0.2 for the wild-type strain. The same experiments carried out with the mutant MD strain, which is resistant to allyl alcohol, led to similar results but with a maximum of 0.84 mol mol(-1) for a glucose/glycerol molar ratio of 0.1. Beyond a molar ratio of 0.2, the biosynthesis of enzymes for the glycerol metabolism was less subject to catabolic repression by glucose in the mutant MD strain than in the wild-type strain.     

Regulation of glucose catabolism in Thiobacillus A2 grown in the chemostat under dual limitation by succinate and glucose

In contrast to its diauxic behaviour in batch culture, Thiobacillus A2 grew in chemostat culture using glucose and succinate as dual limiting substrates. Biomass production under dual limitations was the sum of that on single substrates with each substrate being oxidized and assimilated to similar extents in single and dual substrate-limited cultures. In glucose and glucose + succinate-limited cultures glucose was oxidized largely by the Entner-Doudoroff and pentose phosphate pathways, but other mechanisms also contributed and the ratios of pathways depended on substrate ratios and the previous substrate-history of the culture. Variations in specific activities of enzymes of carbohydrate metabolism following switches from single to mixed substrates were considerable, ranging from fourfold for fructose diphosphate aldolase to more than 200-fold for hexokinase, fructose diphosphatase, glucose 6-phosphate and 6-phosphogluconate dehydrogenases. Changes in specific activities occurred only over prolonged time periods in the chemostat, probably reflecting low concentrations of free substrates in carbon-limited cultures and consequent low levels of catabolite repression.     

Mutual adjustment of glucose uptake and metabolism in Trypanosoma brucei grown in a chemostat.

The mutual adjustment of glucose uptake and metabolism in the insect stage of the protozoan parasite Trypanosoma brucei was studied. T. brucei was preadapted in the chemostat to conditions in which either glucose or proline served as the major carbon and energy source. Cells were grown and adapted to either energy or non-energy limitation at a low dilution rate (0.5 day-1) or a high dilution rate (1 day-1). The cells were then used in short- to medium-term uptake experiments with D-[14C]glucose as a tracer. In time course experiments a steady state was reached after 15 min regardless of the preadaptation conditions. This steady-state level increased with increasing glucose availability during preadaptation. The rate of glucose uptake and the hexokinase activity were linearly correlated. In short-term 5- to 90-s) uptake experiments a high transport rate was measured with cultures grown in excess glucose, an intermediate rate was measured with proline-grown cultures, and a low rate was measured in organisms grown under glucose limitation. Glucose metabolism and proline metabolism did not affect each other during the 15-min incubations. Glucose uptake, as a function of the external glucose concentration, did not obey simple Michaelis-Menten kinetics but could be described by a two-step mechanism: (i) transport of glucose by facilitated diffusion and (ii) subsequent metabolism of glucose. The respective rates of the two steps were adjusted to each other. It is concluded that T. brucei is capable of adjusting the different metabolic processes in a way that gives maximum energy efficiency at the cost of short-term flexibility.     

Isolation of Hanseniaspora uvarum (Kloeckera apiculata) in humans

Isolation of Hanseniaspora uvarum, a yeast of the ascomycetes group, whose anamorph corresponds to Kloeckera apiculata, obtained from stool and two ungual specimens from three patients, is reported. This yeast has been found in soil, water, various fruits, bivalve molluscs, crabs, prawns and fruit flies; in Spain, it has been described in the fermentation processes of some wines. In our region, it has also been found in the intestine of mackerel ( Scomber scombrus). Its finding in humans constitutes a clinical rarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Gluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture

The metabolism of gluconate by Klebsiella pneumoniae NCTC 418 was studied in continuous culture. Under all gluconate-excess conditions at low culture pH values (pH 4.5–5.5) the majority (70–90%) of the gluconate metabolized was converted to 2-oxogluconate via gluconate dehydrogenase (GADH), although specific 2-oxogluconate production rates under potassium-limited conditions were significantly lower than under other gluconate-excess conditions. At high culture pH values, metabolism shifted towards production of acetate. Levels of GADH were highest at low culture pH values and synthesis was stimulated by the presence of (high concentrations of) gluconate. An increase in activity of the tricarboxylic acid cycle was accompanied by a decrease in GADH activity in vivo and in vitro, suggesting that the GADH serves a role as an alternative energy-generating system. Anaerobic 2-oxogluconate production was found to be possible in the presence of nitrate as electron acceptor. Levels of gluconate kinase were highest when K. pneumoniae was grown under gluconate-limited conditions. Under carbon-excess conditions, levels of this enzyme correlated with the intracellular catabolic flux.Abbreviations GADH gluconate dehydrogenase (EC 1.1.99.3) - GAK gluconate kinase (EC 2.7.1.12) - GDH glucose dehydrogenase (EC 1.1.99.17) - PQQ pyrroloquinoline quinone [2,7,9-tricarboxy-1-H-pyrrolo (2,3-f) quinoline-4,5-dione] - TCA trichloroacetic acid     

Effects of glucose supply on myeloma growth and metabolism in chemostat culture

The effects of the glucose supply on growth and metabolism of an SP2/0 derived recombinant myeloma cell line were studied in chemostat culture during growth on IMDM medium at a fixed dilution rate of 0.032 h^?1. Lowering of the feed medium glucose concentration from 25.0 to 1.4 mmol/L resulted in a decrease of steady-state viable cell concentration from 1.9 × 10⁹ L^?1, whereas viability remained above 90%. Mass balances indicated that only a minor amount of glucose was utilized via the TCA cycle irrespective of the glucose concentration in the feed medium. The apparent biosynthetic yield of cells from ATP was independent of the ratio between the specific glucose and glutamine consumption rate. It is concluded that the primary role of glucose is the provision of intermediates for anabolic reactions. In addition, glucose may play an indirect catabolic role in the process of glutaminolysis by providing the pyruvate for the transamination of glutamate to alanine and α-ketoglutarate. At low glucose concentrations in the feed medium, glutamine is probably the sole energy source for this myeloma in chemostat culture. © 1995 Wiley-Liss, Inc.     

Molecular and technological approaches to evaluate strain biodiversity in Hanseniaspora uvarum of wine origin

AIMS: The characterization by molecular and physiological methods of wild apiculate strains, isolated from 'Aglianico del Vulture' grape must. METHODS AND RESULTS: The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species. CONCLUSIONS: The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.     

Glucose catabolism by Thiobacillus A2 grown in chemostat culture under carbon or nitrogen limitation

Thiobacillus A2 was grown in glucose- or ammonium-limited chemostats and relative contributions of the Embden-Meyerhof (EM), Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to glucose catabolism estimated by ¹⁴C-glucose radiorespirometry. In fast growing strain GFI, the EM pathway predominated (41–79%) under all growth conditions with the PP pathway contributing 18–30%. The ED pathway was apparently absent under some conditions of glucose limitation. In contrast, wild type Thiobacillus A2 exhibited predominance of the EM pathway (43–48%) under ammonium-limitation but apparent predominance of the PP pathway (43–55%) under glucose-limitation, although all three pathways were calculated to operate. Under some conditions of glucose limitation the EM pathway was possibly considerably depressed. No clear pattern of response of the three pathways to altered environmental conditions could be deduced, although marked change in pathway activities were obviously induced. Growth yield was apparently unaffected by variation in pathways. The problems of interpreting such complex radiorespirometric data are discussed.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - PK phosphoketolase - PP pentose phosphate     

Quantification of multiple-substrate controlled growth-simultaneous ammonium and glucose limitation in chemostat cultures of Klebsiella pneumoniae

In chemostat cultures of Klebsiella pneumoniae (K. aerogenes) NCTC 418 we measured the concentrations of glucose and ammonium and we varied the ratio of the (limiting) concentrations of glucose and ammonium in the feed medium. By doing this at different dilution rates we found a range where growth rate varies with either concentration in the culture when the other concentration in the culture is held constant. This proves that within this range, dual-substrate controlled growth occurs. Dual substrate-controlled growth was accompanied by yield coefficients for glucose and for ammonium that were intermediate between the yield coefficients obtained for single glucose or single ammonium limitation. We quantified the control by either substrate in terms of the flux control coefficient with respect to that substrate, where flux refers to growth rate. Dualsubstrate controlled growth is reflected by the finding that both flux control coefficients exceed zero, simultaneously. In the transition of glucose to ammonium limitation, the control gradually shifts from glucose to ammonium.Abbreviations Symbol Units Meaning s Steady-state concentration of substrate in the culture - S_r M Concentration of substrate in reservoir medium - Y gDWmol^-1 Yield - D h^-1 Dilution rate - h^–1 Specific growth rate - _max h^–1 Maximal growth rate - C ₂ Control coefficient, of s on - J h^-1 Rate or flux - J_ATP mmolgDW^-1h^-1 ATP synthesis rate - a Anabolism - c Catabolism - l Leak     

Purification and properties of an alcohol dehydrogenase (HUADHII) from Hanseniaspora uvarum K5

An alcohol dehydrogenase HUADHII was purified 43.2-fold from Hanseniaspora uvarum K₅. The enzyme was trimeric with subunits of mol. wt 42 kDa. The N -terminal amino acid sequence of HUADHII has between 45 and 75% identity with part of the sequence of isoenzymes related to group I from Saccharomyces cerevisiae and Kluyveromyces lactis. C2–C4 alcohols and aldehydes were the preferred substrates. The presence of an'α'double bond increased the enzyme activity both for alcohols and aldehydes. It was significantly inhibited by metal-binding agents and thiol reagents. Kinetic studies suggested that HUADHII catalyses the oxidation of ethanol by a random sequential mechanism. It appears that HUADHII, a cytoplasmic fermentative enzyme, is structurally and functionally similar to members of the group I alcohol dehydrogenases.     

Microcalorimetric investigations of the metabolism of yeasts. Growth in chemostat cultures on glucose

Summary As part of a project on the production of penicillin, the penicillin production of two strains of Penicillium chrysogenum which have a different penicillin productivity was investigated in bubble column bioreactors and for comparison in stirred fermenters. The main interest of this study were the complicated interrelations between the stirrer speed, the stirrer type, the shear stress, the morphology of the mycelium and broth viscosity as well as the effect of the oxygen transfer behavior on antibiotic productivity.Stirred tank reactors with different turbine stirrers as well as with a draught tube and propeller were employed.The main variable investigated was the stirrer speed. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply of pulpy mycelia is improved and more cell mass is formed. This result is the same for both strains in all three reactors.If the oxygen partial pressure is near the lower cirtical value, a high percentage of the carbon source is converted into penicillin but the penicillin productivity is low due to a low percentage of penicillin producing cells. At oxygen partial pressures just above 8% saturation, the absolute penicillin productivity is maximal. At higher stirrer speeds and dissolved oxygen concentrations the penicillin production phase is shorter, cell growth is higher and a higher percentage of the carbon source is converted into CO₂.In reactors with a draught tube and propeller, a lower productivity is attained than in those with turbine stirrers.The behavior of the two strains is fairly similar. The higher producing strain, however, has a more distinct separation between its periods of growth and production than does the low producing one. At high stirrer speeds the increase in the cell growth rate is less significant and the substrate yield coefficients are higher for the high producing strain than for the low producing one.Symbols C Dissolved oxygen concentration (mg l^–1) - C^* C at saturation (mg l^–1) - k_La Volumetric mass transfer coefficient (h^–1) - OTR Oxygen transfer rate (mg l^–1 h^–1) - OUR Oxygen uptake rate (mg l^–1 h^–1) - rpm Impeller speed (min^–1) - X (Dry) biomass concentration (g kg^–1) - V_g Volumetric gas flow rate (Nl min^–1) - CMC Carboxymethyl cellulose     

Regulation of the tricarboxylic acid cycle and poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii grown under nitrogen or oxygen limitation.

Azotobacter beijerinckii was grown in ammonia-free glucose/mineral salts media in chemostat culture under oxygen or nitrogen limitation. Selected enzymes of the tricarboxylic acid cycle and poly-beta-hydroxybutyrate metabolism were monitored in relation to oxygen supply for both steady and transition states. Two dissolved oxygen concentrations were used for the nitrogen-limited steady state to investigate the possible effects of respiratory protection of nitrogenase on these enzymes. The levels of NADH oxidase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase increased markedly on relaxation of oxygen limitation while pyruvate dehydrogenase and citrate synthase were relatively unaffected. beta-Ketothiolase and acetoacetyl-CoA reductase levels decreased as oxygen limitation was relaxed. Respiratory activity, as measured by the QO2 value, increased with oxygen supply rate. Imposition of oxygen limitation on a nitrogen-limited culture caused an immediate increase in the NADH/NAD ratio but this rapidly readjusted to its previous steady-state value. These changes are discussed in relation to respiratory protection of nitrogenase and poly-beta-hydroxybutyrate metabolism in A. beijerinckii.     

Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.     

Oxygen and/or glucose limitation in a chemostat culture of Candida utilis mathematical model identification

The glucose and/or dissolved oxygen limited continuous culture of yeast Candida utilis was studied. Six different mathematical models were designed to describe and analyze the experiments. The model considering the production of surface active compounds at autoanaerobic conditions and dissolved oxygen consumption for nongrowth associated exogeneous respiration yields the best fit. The results may be applied for aerobic waste water treatment plants, process analysis and simulation.     

Influence of redox potential on the glucose catabolism of chemostat grownBacillus licheniformis

Summary The effect of the redox potential on the by-production of acetic acid byBacillus licheniformis grown in a chemostat has been studied. It was demonstrated thatB. licheniformis produced acetic acid when it was grown at a low growth rate, low oxygen supplies, and low redox potentials.Furthermore, it was shown that the acetic acid productivity was strongly dependent on redox potential, giving a maximum productivity at redox potentials about 110 mV. The observed effect was correlated to the established theory of the dependence of intracellular enzymes on oxygen and redox potential. It is proposed that the redox potential can be a variable that can be used in a regulatory procedure.     

Competition between oral Streptococcus species in the chemostat under alternating conditions of glucose limitation and excess

Abstract Oral Streptococcus species experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. To investigate the competitiveness of various streptococcal species under fluctuating carbohydrate supply, 2-membered chemostat cultures were run.
Under continuous limitation of glucose or sucrose, all 6 Streptococcus mutans test strains were outcompeted by Streptococcus sanguis P4A7 or Streptococcus milleri B448. This indicated that S. mutans had a lower affinity for glucose and sucrose than S. sanguis and S. milleri .
Mixed cultures were then subjected to hourly pulses with glucose. Under these conditions S. mutans Ny344 competed successfully with S. milleri B448, but still lost the competition against S. sanguis P4A7. The streptococci responded to pulses by taking up glucose at the maximum rate almost instantaneously. S. sanguis P4A7 had the highest rate of glucose uptake while the q _max value of S. mutans Ny344 was higher than that of S. milleri B448. This suggested a causal relationship between q _max and competitiveness.     

Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

Klebsiella pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-limited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.     

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.     

Evaluation of Saccharomyces cerevisiae grown in a multistage chemostat environment under increasing levels of glucose

The concept of net consumption and/or production rates of fermentation metabolites has been proposed and applied to evaluate the performance of Saccharomyces cerevisiae grown in a multistage chemostat environment under increasing glucose concentrations (increased gravity). Results show that both rates are strongly affected by high glucose doses (osmotic pressure) and that ethanol inhibition has a profound influence on the performance of yeast cells.