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Chemostat Cultures (chemostat + culture)
Selected AbstractsInfluence of phenanthrene and fluoranthene on the degradation of fluorene and glucose by Sphingomonas sp. strain LB126 in chemostat culturesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2003René van Herwijnen Abstract Since bacteria degrading polycyclic aromatic hydrocarbon compounds (PAHs) in polluted soils are generally exposed to mixtures of PAHs, we examined the influence of simple PAH mixtures on the degradation activity of Sphingomonas sp. strain LB126. Fluorene serves as sole carbon and energy source for the strain LB126 and phenanthrene and fluoranthene are cometabolically degraded by this species. Chemostat cultures of the strain LB126 were used to study a potential inhibiting effect of phenanthrene and fluoranthene on the degradation of fluorene that was previously observed in batch cultures. We also looked at the effect of phenanthrene on the degradation of glucose in a chemostat culture to see if this effect was specific for the PAH-metabolic pathway or for the total metabolism of the strain. The co-substrates were supplied in a 5% to 30% fraction of fluorene. Phenanthrene and fluoranthene had no significant influence on growth. However, fluorene degradation was inhibited by both phenanthrene and fluoranthene. The effect of phenanthrene was about 10 times stronger than the effect of fluoranthene. Nevertheless, more than 95% removal of fluorene took place together with more than 95% removal of either phenanthrene or fluoranthene. The effect of phenanthrene on the strain LB126 could be ascribed to both toxicity and competitive inhibition, but the effect observed at steady state was due to competitive inhibition only. It appeared that the strain LB126 adapts to the toxicity of phenanthrene within five generations. The inhibitory effects observed previously in batch cultures of the strain LB126 should mainly be ascribed to the toxic effect of phenanthrene. [source] Separation and characterization of the 1,3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild-type and mutant DJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2001H. Malaoui H. MALAOUI AND R. MARCZAK. 2001. Aims:,Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. Methods and Results:,These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. Conclusion:,The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. Significance and Impacts of the study: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol. [source] Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuniENVIRONMENTAL MICROBIOLOGY, Issue 3 2010Edward Guccione Summary Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ,-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni -specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment. [source] Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediationENVIRONMENTAL MICROBIOLOGY, Issue 5 2008Regina A. O'Neil Summary Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments. [source] Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulphate and tetrachloroetheneENVIRONMENTAL MICROBIOLOGY, Issue 2 2001Oliver Drzyzga A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a ,biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium. [source] Bioenergetics of the formyl-methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicusFEBS JOURNAL, Issue 1 2003Linda M. I. De Poorter The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2 -dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures (,pH2). Formyl-methanofuran synthesis is an endergonic reaction (,G°, = +16 kJ·mol,1). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (,G,) were found to range between +10 and +35 kJ·mol,1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions. Heterodisulfide reduction (,G°, = ,40 kJ·mol,1) was associated with free energy changes as high as ,55 to ,80 kJ·mol,1. The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow pH2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations. [source] Influence of phenanthrene and fluoranthene on the degradation of fluorene and glucose by Sphingomonas sp. strain LB126 in chemostat culturesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2003René van Herwijnen Abstract Since bacteria degrading polycyclic aromatic hydrocarbon compounds (PAHs) in polluted soils are generally exposed to mixtures of PAHs, we examined the influence of simple PAH mixtures on the degradation activity of Sphingomonas sp. strain LB126. Fluorene serves as sole carbon and energy source for the strain LB126 and phenanthrene and fluoranthene are cometabolically degraded by this species. Chemostat cultures of the strain LB126 were used to study a potential inhibiting effect of phenanthrene and fluoranthene on the degradation of fluorene that was previously observed in batch cultures. We also looked at the effect of phenanthrene on the degradation of glucose in a chemostat culture to see if this effect was specific for the PAH-metabolic pathway or for the total metabolism of the strain. The co-substrates were supplied in a 5% to 30% fraction of fluorene. Phenanthrene and fluoranthene had no significant influence on growth. However, fluorene degradation was inhibited by both phenanthrene and fluoranthene. The effect of phenanthrene was about 10 times stronger than the effect of fluoranthene. Nevertheless, more than 95% removal of fluorene took place together with more than 95% removal of either phenanthrene or fluoranthene. The effect of phenanthrene on the strain LB126 could be ascribed to both toxicity and competitive inhibition, but the effect observed at steady state was due to competitive inhibition only. It appeared that the strain LB126 adapts to the toxicity of phenanthrene within five generations. The inhibitory effects observed previously in batch cultures of the strain LB126 should mainly be ascribed to the toxic effect of phenanthrene. [source] RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression in Streptococcus mutansFEMS MICROBIOLOGY LETTERS, Issue 1 2004Christopher M. Browngardt Abstract Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf. [source] Transcriptional responses of Saccharomyces cerevisiae to preferred and nonpreferred nitrogen sources in glucose-limited chemostat culturesFEMS YEAST RESEARCH, Issue 4 2007Viktor M. Boer Abstract Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae grown with six different nitrogen sources were subjected to transcriptome analysis. The use of chemostats enabled an analysis of nitrogen-source-dependent transcriptional regulation at a fixed specific growth rate. A selection of preferred (ammonium and asparagine) and nonpreferred (leucine, phenylalanine, methionine and proline) nitrogen sources was investigated. For each nitrogen source, distinct sets of genes were induced or repressed relative to the other five nitrogen sources. In total, 131 such ,signature transcripts' were identified in this study. In addition to signature transcripts, genes were identified that showed a transcriptional coresponse to two or more of the six nitrogen sources. For example, 33 genes were transcriptionally upregulated in leucine-grown, phenylalanine-grown and methionine-grown cultures; this was partly attributed to the involvement of common enzymes in the dissimilation of these amino acids. In addition to specific transcriptional responses elicited by individual nitrogen sources, their impact on global regulatory mechanisms such as nitrogen catabolite repression (NCR) were monitored. NCR-sensitive gene expression in the chemostat cultures showed that ammonium and asparagine were ,rich' nitrogen sources. By this criterion, leucine, proline and methionine were ,poor' nitrogen sources, and phenylalanine showed an ,intermediate' NCR response. [source] Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat culturesFEMS YEAST RESEARCH, Issue 8 2006Jan-Maarten A. Geertman Abstract Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h,1) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high kM for formate at low intracellular NAD+ concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol,1 glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes. [source] A new physiological role for Pdr12p in Saccharomyces cerevisiae: export of aromatic and branched-chain organic acids produced in amino acid catabolismFEMS YEAST RESEARCH, Issue 6 2006Lucie A. Hazelwood Abstract Saccharomyces cerevisiae can use a broad range of compounds as sole nitrogen source. Many amino acids, such as leucine, tyrosine, phenylalanine and methionine, are utilized through the Ehrlich pathway. The fusel acids and alcohols produced from this pathway, along with their derived esters, are important contributors to beer and wine flavor. It is unknown how these compounds are exported from the cell. Analysis of nitrogen-source-dependent transcript profiles via microarray analysis of glucose-limited, aerobic chemostat cultures revealed a common upregulation of PDR12 in cultures grown with leucine, methionine or phenylalanine as sole nitrogen source. PDR12 encodes an ABC transporter involved in weak-organic-acid resistance, which has hitherto been studied in the context of resistance to exogenous organic acids. The hypothesis that PDR12 is involved in export of natural products of amino acid catabolism was evaluated by analyzing the phenotype of null mutants in PDR12 or in WAR1, its positive transcriptional regulator. The hypersensitivity of the pdr12, and war1, strains for some of these compounds indicates that Pdr12p is involved in export of the fusel acids, but not the fusel alcohols derived from leucine, isoleucine, valine, phenylalanine and tryptophan. [source] Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous culturesFEMS YEAST RESEARCH, Issue 2 2003Helena Albergaria Abstract The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures. By both cultivation techniques this yeast was found to be Crabtree-positive. Up to a dilution rate of 0.25 h,1, glucose was completely metabolised into biomass, glycerol and carbon dioxide. Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids. Biomass yield during the purely oxidative growth was 0.49 g g,1 and decreased to 0.26 g g,1 for D=0.34 h,1. A maximal specific ethanol production rate of 1.36 mmol g,1 h,1 and a maximal ethanol yield of 0.05 g g,1 were achieved at D=0.34 h,1. [source] Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2002Jasper A. Diderich Abstract In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate. [source] Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor inductionBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2010Beum Jun Kim Abstract The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086,h,1 with an initial cell mass of less than 0.6,OD600. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9,OD600 or higher, or at low dilution rates (<0.05,h,1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron-supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6,h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron-limited chemostat cultures was observed compared to iron-supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955,964. © 2009 Wiley Periodicals, Inc. [source] Rapid media transition: An experimental approach for steady state analysis of metabolic pathwaysBIOTECHNOLOGY PROGRESS, Issue 1 2010Hannes Link Abstract Commonly steady state analysis of microbial metabolism is performed under well defined physiological conditions in continuous cultures with fixed external rates. However, most industrial bioprocesses are operated in fed-batch mode under non-stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup,rapid media transition,enables steady state perturbation of metabolism on a time scale of several minutes in parallel to operating bioprocesses. For this purpose, cells are separated from the production process and transferred into a lab-scale stirred-tank reactor with modified environmental conditions. This new approach was evaluated experimentally in four rapid media transition experiments with Escherichia coli from a fed-batch process. We tested the reaction to different carbon sources entering at various points of central metabolism. In all cases, the applied substrates (glucose, succinate, acetate, and pyruvate) were immediately utilized by the cells. Extracellular rates and metabolome data indicate a metabolic steady state during the short-term cultivation. Stoichiometric analysis revealed distribution of intracellular fluxes, which differs drastically subject to the applied carbon source. For some reactions, the variation of flux could be correlated to changes of metabolite concentrations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Exploration of the hydrogen producing potential of Rhodobacter capsulatus chemostat cultures: The application of deceleration-stat and gradient-stat methodologyBIOTECHNOLOGY PROGRESS, Issue 5 2009Sebastiaan Hoekema Abstract In this work, the dependency of the volumetric hydrogen production rate of ammonium-limited Rhodobacter capsulatus chemostat cultures on their imposed biomass concentration and dilution rate was investigated. A deceleration-stat experiment was performed by lowering the dilution rate from 1.0 d,1 to zero aimed at a constant biomass concentration of 4.0 g L,1 at constant incident light intensity. The results displayed a maximal volumetric hydrogen production rate of 0.6 mmol m,3 s,1, well below model predictions. Possibly the high cell density limited the average light availability, resulting in a sub-optimal specific hydrogen production rate. To investigate this hypothesis, a gradient-stat experiment was conducted at constant dilution rate of 0.4 d,1 at constant incident light intensity. The biomass concentration was increased from 0.7 to 4.0 g L,1 by increasing the influent ammonium concentration. Up to a biomass concentration of 1.5 g L,1, the volumetric hydrogen production rate of the system increased according to model predictions, after which it started to decline. The results obtained provide strong evidence that the observed decline in volumetric hydrogen production rate at higher biomass concentrations was at least partly caused by a decrease in light availability. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | |||||||||||||||||||||||||