Growth In Chemostat Research Articles

A structured model describing carbon and phosphate limited growth of Catharanthus roseus plant cell suspensions in batch and chemostat culture

The growth of plant cell suspension cultures of Catharanthus roseus in batch fermentors was studied at different initial phosphate levels of the medium. On the basis of the observations and existing knowledge with respect to phosphate metabolism in cultured C. roseus cells, a structured mathematical model was developed for the description of the kinetics of growth and intracellular accumulation of glucose and phosphate, as a function of glucose and phosphate supply. It was shown that the model offers not only good description of the growth of the cells in batch culture at different initial phosphate levels, but also provided a satisfactory description of the growth in glucose limited chemostats.

Glucose and carbon dioxide metabolism by Succinivibrio dextrinosolvens

Growth rates and culture conditions affect the molar yields of catabolic end products and cells of Succinivibrio dextrinosolvens growing on glucose. When growth in chemostats occurred, a trend toward decreased succinate and acetate formation, increased lactate formation, and a higher yield of cells correlated with an increase in the growth rate. End product and cellular yields on defined medium indicate a high maintenance requirement for S. dextrinosolvens and are consistent with energy conservation steps during the formation of acetate and succinate. Simultaneous carbon dioxide consumption and production were determined from batch studies with NaH14CO3, and the amounts were used to calculate a fermentation balance. These data also indicated that CO2 consumption lags behind CO2 production early in the growth phase, becoming equivalent to it toward stationary phase. Significantly more CO2 was fixed by S. dextrinosolvens when the organism was cultured in chemostats sparged with CO2. Formate is in part derived from free CO2 in the medium, as shown by 13C nuclear magnetic resonance studies, and may be sensitive to CO2 availability. Nuclear magnetic resonance data are consistent with the carboxylation of a C3 intermediate of the Embden-Meyerhof-Parnas pathway of glycolysis to a C4 compound to eventually form succinate.

Kinetics of microbial growth with mixtures of carbon sources

Several investigations have shown that during growth in carbon-limited chemostats the simultaneous utilisation of carbon substrates which usually provoke diauxie under batch conditions, i.e., 'mixed substrate growth,' is probably the rule under ecologically relevant growth conditions. In contrast, the models presently available for the description of the kinetics of microbial growth are all based on the use of single substrates. Systematic studies in chemostat culture have shown that steady-state residual concentrations of individual compounds were consistently lower during mixed substrate growth than during growth with the single substrates. This effect is clearly demonstrated for the case of Escherichia coli growing with mixtures of glucose plus galactose. The data presented indicate that the extent of reduction of steady-state residual substrate concentration is dependent on the proportions of the substrates in the mixture, the nature of substrates mixed and the regulation pattern of enzymes involved in their breakdown. If this behaviour can be shown to be typical for growth under environmental conditions, it may provide an explanation why microbes still grow relatively fast at the low substrate concentrations encountered in nature.

The influence of energy deficiency‐imposing conditions on the capacities ofAcetobacter methanolicus to oxidize glucose and to produce gluconic acid

AbstractThe presence of dinitrophenol (DNP) during the chemostatic growth of Acetobacter methanolicuson glucose led to i) no significant increase in glucose dehydrogenase, ii) a decrease in the capacity to oxidize glucose by about 30%, not depending on the DNP concentration, and iii) a gradual decrease in the capacity to form gluconic acid, depending on the concentration of the uncoupler. Similar effects of DNP on Acetobacter methanolicuswere observed during the gluconic acid formation process. The loss in the capacity to form gluconic acid is indicated by a drop in the ATP concentration of the cells which cannot be counteracted by the ATP syntheses originating from the oxidation of gluconic acid and of glucose. ATP could be necessary for pumping protons out of the cells that were taken up together with gluconic acid and by the effect of DNP.

Effect of 5,7-unsaturated sterols on ethanol tolerance in Saccharomyces cerevisiae.

Structural membrane lipids are known to contribute to the high ethanol resistance of Saccharomyces cerevisiae (2, 4, 17). By manipulating the yeast cellular sterol level by changing the carbon-to-nitrogen source ratio in the chemostat growth medium, high delta 5,7-sterol levels were found to increase the resistance of yeast populations to ethanol-induced death. The resistance of the erg2 (delta 8----delta 7-sterol isomerase) mutant to ethanol-induced death was generally comparable with that of the delta 5,7-sterol-synthesizing strain. In contrast, the sensitivity of anaerobic growth to inhibition by ethanol was higher in the erg2 mutant in comparison with the delta 5,7-sterol-synthesizing strains but a high level of those sterols increased the vulnerability of anaerobic growth to ethanol inhibition.

Selection of laboratory wild-type phenotype from natural isolates of Escherichia coli in chemostats.

We have followed, in glucose-limited chemostats, the evolution of natural isolates of Escherichia coli possessing maximal growth rates of 0.48-1.43 doublings/h. Under these conditions a rapid-growth phenotype similar to that of standard laboratory wild-type strains was selected so that after 280 generations all of the cultures were characterized by bacteria with maximum growth rates close to 1.33 doublings/h. The growth yields of the natural isolates, on the other hand, were quite uniform and improved only slightly during the selection; it seems that the natural isolates are nearly maximally efficient at utilizing glucose. Some of the kinetic characteristics of ribosomes prepared from natural isolates vary markedly and in proportion to the growth rates of the original strains. After growth in glucose-limited chemostats, the ribosomes of all of the cultures become kinetically indistinguishable from those of laboratory wild-type bacteria. These observations confirm the interpretation that bacteria grown under normal laboratory conditions have been selected for maximum growth rates which demand maximum translation efficiency. In contrast, these characteristics do not seem to be strongly selected in the natural isolates.

Regulation and utilization of microsin promoter turn on in a chromosomal fusion

Prior work has demonstrated that the microsin antibiotics are produced by enteric bacteria when the growth medium is depleted of nutrients. Because the control loci could have biotechnical potential, and general stress-response phenomena are of importance to understanding how bacteria survive in natural and bioreactor environments, we examined further the growth rate dependence of gene expression under the control of the microsin B17 promoter. This work entailed performing batch and chemostat growth experiments with a strain of E. coli K-12 containing a mcbA-lacZ gene fusion in the chromosome. Our results indicate that when a culture is presented with excess respiratory substrate, a well defined growth rate exists, below which a significant induction event occurs. However, cultures that are fermenting or highly glycolytic tend to express poorly. Additionally, the utility of the fusion strain was examined by performing fed-batch cultivation experiments. We found that sustained production in a fed-batch reactor can be accomplished by using a straightforward, exponential nutrient feeding profile.

Competition between strains of Escherichia coli with and without plasmid RP4 during chemostat growth

Escherichia coli strains J53(nal) and J53(RP4) were grown together in glucose-limited continuous cultures. Based on the measured growth kinetic constants of the two strains, take-over of the cultures by J53(RP4) was predicted. However, in practice, an initial period of predominance by J53(RP4) was always followed by a prolonged period in which relative numerical proportions of the two strains oscillated widely. This period of oscillation was removed or greatly reduced when the difference between the predicted growth-rate potentials of the two strains was increased by selection of a chemostat-adapted variant of J53(RP4).

Physiological, biochemical, and mathematical studies of micro‐aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. III: Mathematical model of cellular energetics and catabolism

Mathematical models of the catabolic pathways, the utilization and waste of ATP, and the factors affecting yeast growth in a micro-aerobic chemostat are presented. The models incorporate the intracellular metabolite and enzyme activity assays performed in Part II to explain the unusual macroscopic chemostat behaviors reported in Part I. The catabolic model successfully predicts a maximum in the specific ethanol productivity as a function of the intracellular ATP concentration. The ATP balance model enables the prediction of the intracellular ATP concentration and the ATP yield for given dissolved oxygen concentrations. Finally, in the context of a growth model, singularity theory provides a framework to explain the transition observed in Part I between hysteresis and the monotonic biomass versus oxygenation profiles in response to changes in the nutrient composition. The models serve to organize data and to concretely express proposed metabolic mechanisms and cause-effect hypotheses. The model is only applicable to the micro-aerobic and excess glucose conditions encountered in this study.

Studies on saccharomyces cerevisiae cinder carbon-limiting growth transformed with plasmid pcyg4 that carries the gene for NADP-GDH

The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).

Further evidence for the existence of a bottleneck in the metabolism ofSaccharomyces cerevisiae

The growth physiology of Saccharomyces cerevisiae strains H1022 and Whi2+ has been studied in aerobic batch and continuous (chemostat) cultures. Results from the measurement of biomass and medium components (off-line) together with oxygen, carbon dioxide and heat measurements (on-line) have been used in an attempt to explore the existence of 'overflow' or 'bottleneck' metabolism as opposed to catabolite repression (Crabtree effect) in these strains. Chemostat experiments indicated that specific oxygen uptake rate (qO2) was linearly related to the dilution rate (D) at values below the critical dilution rate (D crit), becoming constant above D crit, which is in agreement with the bottleneck theory. However, batch culture experiments indicated negligible oxygen consumption during the initial glucose growth phase, the culture exhibiting purely anaerobic metabolism. The bottleneck theory would propose that qO2 has a constant (maximum) value under these conditions. The results presented here suggest that while the bottleneck theory can be adequately used to describe chemostat growth of S. cerevisiae, some other control mechanism must be operating under conditions of high glucose concentrations, such as those initially prevailing in the batch culture experiments.

The Influence of C:N Ratio in the Growth Medium on the Cellular Composition and Regulation of Enzyme Activity in Hyphomicrobium X

The regulation of the enzymes associated with one-carbon metabolism and the assimilation of nitrogen, together with the cellular composition of Hyphomicrobium X, were investigated. The effect of changing the methanol-carbon concentration with the NH+ 4-nitrogen concentration remaining constant (C: N ratio) in the medium during chemostat growth at a constant dilution rate was studied. As the medium changed from a C-limitation to a dual C- and N- and finally a N-limitation, the culture gradually passed through three definite growth phases. In response to these environmental conditions the cellular composition and the specific enzyme activity patterns changed. The C-content of the cells changed very little. The N- and protein-content was constant over C-limiting conditions, but under dual C- and N-limiting and N-limiting conditions an accumulation of poly-β-hydroxybutyrate (PHB) occurred and as a consequence the N-content and protein-content of the cells decreased. The enzyme associated with N-assimilation during C-limitation was an NADP+-dependent glutamate dehydrogenase which was replaced by the high affinity glutamine synthetase and glutamate synthase pathway immediately the NH+ 4-N concentration in the medium became limiting. Similarly the specific activity of methanol dehydrogenase, which was high during C-limiting conditions, dropped to a low level as the NH+ 4-N concentration decreased. Finally carbon balances were constructed throughout the experiment which showed that irrespective of the C:N ratio in the medium during C-limitation, the methanol-carbon was fluxed into biomass and CO2 only; during dual limitation the carbon was channelled into biomass, CO2 and PHB; and finally when the growth was in the presence of excess carbon no methanol-carbon was directed into over-metabolite production but, instead, the excess carbon was oxidized through the dissimilatory pathway.

Analysis of the variation in ethanol yield from glucose or xylose with continuously grownThermoanaerobacter ethanolicus

The chemostat growth ofThermoanaerobacter ethanolicus was limited by either glucose or xylose. Ethanol yield was dependent primarily upon length of cultivation time and secondarily upon growth rate. Low growth rates favored high ethanol yields (0.42 g/g) from xylose.

The Impact of a Chemostat Discharge Containing Oil Degrading Bacteria on the Biological Kinetics of a Refinery Activated Sludge Process

The effect of discharging specific oil degrading bacteria from a chemostat to a refinery activated sludge process was determined biokinetically. Plant data for the kinetic evaluation of the waste treatment plant was collected before and during treatment. During treatment, the 500 gallon chemostatic growth chamber was operated on an eight hour hydraulic retention time, at a neutral pH, and was fed a mixture of refinery wastewater and simple sugars. The biokinetic constants k (days−1), Ks (mg/L), and K (L/mg-day) were determined before and after treatment by Monod and Lineweaver-Burk plots. Solids discharged and effluent organic concentrations were also evaluated against the mean cell retention time (MCRT). The maximum utilization rate, k, was found to increase from 0.47 to 0.95 days−1 during the operation of the chemostat. Subsequently, Ks increased from 141 to 556 mg/L. Effluent solids were shown to increase slightly with treatment. However, this was acceptable due to the polishing pond and the benefit of increased ability to accept shock loads of oily wastewater. The reason for the increased suspended solids in the effluent was most likely due to the continual addition of bacteria in exponential growth that were capable of responding to excess substrate. The effect of the chemostatic addition of specific microbial inocula to the refinery waste treatment plant has been to improve the overall organic removal capacity along with subsequent gains in plant stability.

Scum actinomycetes in sewage treatment plants—Part 1: Growth kinetics of Nocardia amarae in chemostat culture

The scum actinomycete Nocardia amarae was grown in batch culture and in chemostat culture on a fructose-peptone-yeast extract substrate. Chemostat growth is modeled with a two-substrate Monod equation incorporating 6 parameters which include maximum growth, kinetic constants for substrate and oxygen, cell yield and wall growth. These parameters were identified by numerically optimizing the fit. Among these parameters μ max = 0.087 ± 0.0075 h −1 and K s = 675 ± 124 mg l −1, a value much higher than found for other activated sludge bacteria. The results are compared with findings of other researches for other sludge bacteria. Different growth strategies of these organisms are discussed.

Suboptimal growth with hyper-accurate ribosomes.

Mutant bacteria with hyperaccurate ribosomes support their excessive accuracy of translation in vitro by dissipating 1.5 to 2.5 cognate ternary complexes per peptide bond formed. This is to be compared with a dissipation rate close to 1.1 for wild-type ribosomes. Here, we have tested the hypothesis that a corresponding loss of translational efficiency in vivo would lower the growth rate of the mutants. Such a growth inhibitory effect would explain why the lower accuracy of wild-type ribosomes is more fit. Our data show that as expected the mu of the hyperaccurate mutants is smaller than that of wild-type bacteria. In contrast, during glucose-limited growth in chemostats there is not the same simple correlation between growth yield and ribosomal efficiency for the hyperaccurate mutants.

The relationship between inorganic nitrogen oxidation and organic carbon production in batch and chemostat cultures of marine nitrifying bacteria

Rates of nitrification and organic C production were determined in batch and chemostat cultures of marine nitrifying bacteria; two NH 4 + -oxidizing species and one NO 2 − -oxidizing spezies. With increasing age in batch cultures and with decreasing flow rates in chemostats, cellular organic C and N concentrations declined while the intracellular ratio of C:N remained constant. With decreasing flow rates in chemostats, there was a reduction in (a) carboxylating enzyme activity per unit of cellular organic C (the potential for chemoautotrophic CO2 fixation), and (b) the yield of organic C. For both NH 4 + and NO 2 − oxidizers, rates of nitrification and C yield were lowest at very slow chemostat growth rates, when compared with optimal growth rates in batch cultures. For both NH 4 + and NO 2 − -oxidizing species, the stoichiometric relationship between nitrification and organic C production did not remain constant and appeared to be dependent on the availability of the inorganic N substrate. The organic C yield from NH 4 + oxidation and hence the free energy efficiency declined with increasing age in batch cultures and with decreasing flow rates in chemostats. The C yield from NO 2 − oxidation and the free energy efficiency at slow chemostat growth rates was also lower than that at the optimal growth rate in batch culture.

Stability of a Catabolic Plasmid in Continuous Culture

A wild-type strain of Pseudomonas putida PPK1, carrying a non-conjugative TOL plasmid, was grown in continuous culture under carbon-limitation with m-toluate as growth substrate. When the medium was changed to benzoate a plasmid-free strain appeared after about 100 h. After this event the proportion of plasmid-containing bacteria declined rapidly but in several experiments this was followed by oscillations in the relative frequencies of the plasmid-free and plasmid-containing populations. About 1% of the total population retained the plasmid after 600 h growth under benzoate-limitation. When the input medium was returned to m-toluate the plasmid-free bacteria disappeared and the TOL+ population recovered to 100%. The plasmid in the wild-type strain was stably maintained during 500 h of chemostat growth under succinate-limitation. TOL+ strains re-isolated from continuous culture under benzoate-limitation retained their TOL plasmids for longer periods. It is suggested that plasmid loss is related to a failure in the control of partitioning at cell division but that this is not absolute and allows the maintenance of a residual low level of plasmid-containing bacteria in the population.

Control of synthesis of wall teichoic acid during balanced growth of Bacillus subtilis W23.

Enzymes involved in the synthesis of teichoic acid and its linkage to the wall in Bacillus subtilis W23 were measured in chemostat cultures growing at equilibrium at a dilution rate of 0.2 h-1 in different concentrations of inorganic phosphate. All the enzymes, except teichoic acid glucosyl transferase, which was insensitive to changes in phosphate concentration, were almost undetectable at 0.5 mM-phosphate. At higher phosphate concentrations the changes in activity of the enzymes of linkage unit synthesis were sufficient to account for the changes in the rate of incorporation of teichoic acid into the wall in vivo. Between 3.5 and 4.5 mM-phosphate the amount of teichoic acid synthesized in vivo increased, but no increase in the ability of toluenized bacteria to synthesize teichoic acid could be detected. Allosteric regulation might therefore be important at high phosphate concentrations. Bacteria maintained a constant ATP content and a constant adenylate energy charge during chemostat growth at all phosphate concentrations.

Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus.

Alcaligenes eutrophus did not form the key enzymes of autotrophic metabolism, the soluble and particulate hydrogenases and ribulosebisphosphate carboxylase (RuBPC), during heterotrophic growth on succinate in batch cultures. During succinate-limited growth in a chemostat, high activities of both hydrogenases were observed. With decreasing dilution rate (D) the steady-state hydrogenase activity (H) followed first-order kinetics, expressed as follows: H = Hmax .e-alpha.D. An identical correlation was observed when autotrophic growth in a chemostat was limited by molecular hydrogen. During autotrophic growth under oxygen or carbon dioxide limitation, the activity if the soluble hydrogenase was low. These data suggested that hydrogenase formation depended on the availability of reducing equivalents to the cells. RuBPC activities were not correlated with the hydrogenase activities. During succinate-limited growth, RuBPC appeared at intermediate activities. During autotrophic growth in a carbon dioxide-limited chemostat, RuBPC was highly derepressed. RuBPC activity was not detected in cells that suffered from energy limitation with a surplus of carbon, as in a heterotrophic oxygen-limited chemostat, nor was it detected in cells limited in carbon and energy, as in the case of complete exhaustion of a heterotrophic substrate. From these data I concluded that RuBPC formation in A. eutrophus depends on two conditions, namely, carbon starvation and an excess of reducing equivalents.