Showing papers on "Chemostat published in 1990"

Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

Abstract: 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 031 h-1 and a Ks for glucose of 055 mM At a dilution rate of 010 h-1, a maximal yield of 010 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 010 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

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts.

Abstract: The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.

Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures.

Abstract: The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production. The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium. Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1. This is much higher than previously reported for batch cultures. Addition of acetic acid or propionic acid lowered the YATP. A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added. This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.

Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle

Abstract: A novel method of cell culture, enabling growth rate control of sessile Gram-negative populations, has been employed to assess the sensitivity of Escherichia coli towards the aminoglycoside antibiotic, tobramycin. Changes in sensitivity, dependent on the growth rate, were compared with those for suspended populations grown in a chemostat and also those for newly-formed daughter cells shed from the biofilm during its growth and development. At specific growth rates up to 0.3 h-1 the susceptibility both of the resuspended biofilm cells and of their planktonic, chemostat grown controls increased in proportion to the growth rate. As the growth rate was increased further (up to 0.7h-1), the susceptibility of the resuspended biofilm cells remained high, whilst that of the planktonic controls decreased. Newly-formed daughter cells, dislodged from the biofilm, demonstrated a uniformly high sensitivity to the antibiotic at all growth rates. This sensitivity corresponded to that of the fastest-growing cells resuspended from biofilms. Lack of growth rate dependency of killing for the newly-formed daughter cells and their high sensitivity to tobramycin suggested that tobramycin activity might vary during the cellular division cycle. Indeed, when synchronous populations were exposed to tobramycin at various times during their division cycle, sensitivity decreased markedly 20 min before the onset of septation, and increased as septation began. Regulation of the cellular division cycle might therefore account, at least partly, for the observed effects of growth rate on susceptibility.

Possible involvement of the division cycle in dispersal of Escherichia coli from biofilms.

Abstract: Growth rate control of adherent, sessile populations was achieved by the controlled perfusion of membrane-associated bacterial biofilms by the method of Gilbert et al. (P. Gilbert, D. G. Allison, D. J. Evans, P. S. Handley, and M. R. W. Brown, Appl. Environ. Microbiol. 55:1308-1311, 1989). Changes in cell surface hydrophobicity were evaluated with respect to growth rate for such sessile Escherichia coli cells and compared with those of suspended (planktonic) populations grown in a chemostat. Newly formed daughter cells shed at the various growth rates from the biofilm during its growth and development were also included in the study. Surface hydrophobicity decreased with growth rate similarly for both planktonic and sessile E. coli; no significant differences were noted between the two. Daughter cells dislodged from the biofilm, however, were significantly more hydrophilic than those remaining, indicating that hydrophobicity changed during the division cycle. Our data support the hypothesis that dispersal of cells from adhesive biofilms and recolonization of new surfaces reflect cell-cycle-mediated events.

Energetics of syntrophic ethanol oxidation in defined chemostat cocultures

Abstract: The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Ymax and me were determined from the total molar growth yields determined at growth (dilution) rates between 0.02 and 0.14 h-1. The individual growth yields of the partner organisms were determined from their numbers and cellular mass in the chemostat cocultures. The Gibbs free energy (ΔG=-16.3 kJ/mol ethanol) available to P. acetylenicus as well as its Ymax (1.7–2.2 g/mol ethanol) were almost constant in the different cocultures. P. acetylenicus shared 44–67% of the total biomass produced, whereas it shared only 19, 23, and 37% of the total Gibbs free energy (ΔG) available from ethanol oxidation coupled to sulfate reduction, methanogenesis, and homoacetogenesis, respectively. The residual 63–81% of the total available ΔG were shared by the H2 oxidizers which exhibited Ymax values being highest for A. woodii (6.6 g/mol acetate) > D. desulfuricans (3.8 g/mol sulfide) > M. bryantii (2.2 g/mol CH4). The results are discussed with respect to ATP generation and coupling of catabolism with cell production.

Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen‐limiting conditions

Abstract: Defined mixed cultures of an obligately aerobic Pseudomonas testosteroni and anaerobic Veillonella alcalescens strain were grown under oxygen and lactate limitation in chemostats with different oxygen supply rates. The aerobic and the anaerobic bacteria were shown to coexist and to complete for common substrates over a wide range of oxygen supply rates. Under similar conditions but with formate as the major substrate chemostat enrichments gave rise to undefined mixed cultures of aerobic, fermentative and methanogenic bacteria. The relevance of these observations to natural mineralization processes is discussed.

Energetics of syntrophic ethanol oxidation in defined chemostat cocultures. 2: Energy sharing in biomass production

Abstract: The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Y max and m e were determined from the total molar growth yields determined at growth (dilution) rates between 0.02 and 0.14 h −1 . The individual growth yields of the partner organisms were determined from their numbers and cellular mass in the chemostat cocultures

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture.

Abstract: The growth and metabolism of Saccharomyces cerevisiae was studied in steady-state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady-state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady-state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro-aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.

A model for energy-sufficient culture growth.

Abstract: Pirt's maintenance model has been widely accepted for the effects of growth rate and maintenance on growth yield. However, the interpretation of parameters in Pirt's model as biological constants is difficult for energy-sufficient culture growth. In this study, a mechanistic model for the growth energetics of energy-sufficient chemostat cultures is proposed and verified with literature data. In the model, the overutilization of the energy substrate in energy-sufficient culture growth is attributed to the defective regulation of the energy substrate metabolism and energy uncoupling. The model also uses an "energy surplus" concept to collectively represent the effects of energy excessiveness. The proposed model provides a better quantitative understanding of the maximum growth yield and maintenance of energy-sufficient cultures. It also explains the glucose concentration effect reported in the literature.

Influence of growth rate on the accumulation of ergosterol in yeast-cells

Abstract: The influence of growth rate on the accumulation of ergosterol inSaccharomyces cerevisiae was studied with glucose, maltose, ethanol and acetic acid as substrates under C- and N-limitations in chemostat experiments. In carbon limited cultures an decrease in ergosterol content with rising dilution rate was observed, whereas in nitrogen limited cells an quite opposite behaviour was attained. A maximum specific rate of ergosterol synthesis of about 2 mg per h per g dry cell mass was calculated for nitrogen limited cultures.

On the coexistence of competing microbial species in a chemostat under cycling.

Abstract: It is known that two microbial species competing for a single rate-limiting nutrient cannot grow together in a chemostat under steady-state operation, but eventually the species with the lower specific growth rate at the particular operating conditions will become extinct. Coexistence of the two populations has been shown obtainable in chemostats under periodic operation. This is possible in cases where the specific growth rate functions of the two species are such that for certain values of the nutrient concentration the first species grows faster than the second, and for other values of the nutrient concentration the second species is the one growing faster. In a previous article it was demonstrated that, even in cases where the specific growth rate functions of the two species are such that one of the species grows faster than the other for all values of the nutrient concentration, extinction of either species is possible provided that time delay in the response of the species to changes in their fermentation environment is accounted for, and that the faster growing species is also faster in its response. Here, we show that coexistence of the two species is also possible in a significant range of the operating parameters. We develop a numerical algorithm with which we trace the boundary of the coexistence region in the entire operating parameter space and construct the operating diagram of the system.

Determination of growth and coupled nitrification/denitrification by immobilized Thiosphaera pantotropha using measurement and modeling of oxygen profiles

Abstract: An oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilized Thiosphaera pantotropha. This organism can convert ammonia completely to nitrogen gas under aerobic conditions (coupled nitrification/denitrification) and denitrifies nitrate at highest rates under anaerobic conditions. Immobilization of T. pantotropha can result in aerobic and anaerobic zones inside the biocatalyst particle which will be advantageous for the conversion of ammonia and nitrate from wastewater. However, information of the effects of immobilization on the physiology of T. pantotropha is necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures of T. pantotropha so that it can be used for immobilized cells. The original model was based on metabolic reaction equations. Kinetic and diffusion equations have now been added. Experimental verification was carried out using a stirred tank reactor and a Kluyver flask. After immobilization in agarose, the cells were grown in the particles under continuous culture conditions for 3 days. After 24 h the oxygen penetration depth showed a constant value of 100 μ, indicating that a steady state was reached. Scanning electron micrographs showed that large colonies of cells were present in this 100-μm aerobic layer.From the dynamics of the start-up phase, several parameters were determined from measurements of the oxygen concentration profiles made every few hours. The profiles simulated by the model were fitted to the measured data. The average value for the maximum specific growth rate was 0.52 h−1, and the maximum oxygen conversion rate was 1.0 mol Cmol−1 h−1. The maximum specific acetate uptake rate was 2.0 mol Cmol−1 h−1, and the Monod constant for acetate was 2.9 × 10−2 mol m−3. The maximum specific nitrification rate was 0.58 × 10−1 mol Cmol−1 h−1, and the amount of oxygen necessary for nitrification was 11% of the total oxygen uptake rate. Most of the kinetic parameters determined for the immobilized cells were in good agreement with those for the suspended cells. Only the maximum specific growth rate was significantly higher, and the maximum specific nitrification rate was some what lower than for suspended cells. The experimental results clearly show that an oxygen microsensor, in combination with mathematical modeling, can successfully be used to elucidate the kinetic behavior of immobilized, oxygen-consuming, cells. (Less)

Shear Sensitivity of Hybridoma Cells in Batch, Fed-Batch, and Continuous Cultures

Abstract: Previously, we observed that CRL-8018 hybridoma cells were more sensitive to well-defined viscometric shear during the lag and stationary phases than during the exponential phase of batch cultures. Some potential hypotheses for explaining the increase in shear sensitivity are (1) nutrient limitations that result in a decrease in production of specific cellular components responsible for the mechanical strength of the cell, (2) nutrient limitations that lead to synchronization of the culture in a cell cycle phase that is more sensitive to shear, or (3) a link between cell growth and shear sensitivity, such that slowly growing cells are more sensitive to shear. Here, the duration of the exponential phase was increased with use of fed-batch, and the effect on shear sensitivity of the cultures was measured with a viscometric technique. Extension of exponential growth resulted in an increased period during which the cells were insensitive to shear. Additionally, the shear sensitivity of the cells was constant over a wide range of growth rates and metabolic yields in chemostat cultures. These observations suggest that as long as the cells are actively (exponentially) growing, their shear sensitivity does not depend on the growth rate or metabolic state of the cell as expressed by metabolic yields. Thus, hypothesis 3 above can be dismissed.

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Abstract: Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 μM pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Ymax and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol−1 and 0.10 mmol · g−1 · h−1, respectively. Neither the Ymax nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.

The selection of microbial communities by constant or fluctuating temperatures

Abstract: The diversity of bacterial communities isolated from Antarctic lake sediment in chemostats under constant low temperature (8°C) or diurnally fluctuating temperature (1°C to 16°C) was examined. The median optimum temperature for growth of the freshwater bacteria isolated from the fluctuation chemostat was significantly lower (P < 1%) than that for those from the constant temperature chemostat. The diversity of the enriched bacterial community isolated in the chemostat culture subjected to short-term temperature fluctuations was greater than that enriched under constant temperature. At least 4 different groups of bacteria, that occupied separate ‘temperature niches’, were isolated from the fluctuating chemostat compared to only one group isolated from the stable chemostat. Furthermore, a pseudomonad from the fluctuating chemostat was shown to out-compete another pseudomonad from the stable chemostat when both were subjected to the fluctuating temperature regime. However, the pseudomonad of constant (8°C) temperature origin out-competed that isolated under fluctuating conditions when subjected to a stable temperature regime.

Controlled growth of competing species

Abstract: A mathematical model of a chemostat, treated as a control process, is considered. Several microorganisms compete for a single essential, growth-limiting nutrient. Earlier theory (supported by experiments) predicts that if the nutrient input and washout rates are constant, at most one competitor survives, and that if either the nutrient input rate or the washout rate is periodic, coexistence is possible under certain conditions. Here, the rate of input of the nutrient is used as the control function. Necessary as well as sufficient conditions for the existence of admissible controls producing coexistence of all the species, and sufficient conditions for the existence of admissible controls for selective extinction of some species, are derived.

A process for the production of human proinsulin in Saccharomyces cerevisiae.

Abstract: In order to develop a large-scale fermentation process for the production of human proinsulin in yeast, the intra-cellular expression of a human superoxide dismutase-human proinsulin fusion product (SOD-PI) has been studied. The expression of SOD-PI in Saccharomyces cerevisiae is regulated by a hybrid alcohol dehydrogenase 2/glyceraldehyde-3-phosphate dehydrogenase promoter. The promoter is repressed by glucose and derepressed by depletion of glucose. Although the genetic stability of the construction is shown to be poor under product-inducing conditions, it is demonstrated in shake flask experiments that a stable expression potential can be maintained in a complex medium for more than 60 generations by maintaining excess glucose throughout the cultivations. These results have been confirmed in continuous cultures in chemostat and turbidostat experiments. Addition of the glucose analogs glucosamine, 2-desoxyglucose, methylglucose, and thioglucose also leads to repression of SOD-PI formation. The analogs, however, are not suitable for improving genetic stability during propagation because of growth inhibition. In batch fermentation experiments in a complex medium at 30 degrees C, it has been demonstrated that initial glucose concentrations up to 50 g/L result in high specific SOD-PI yields giving an overall yield of up to 700 mg SOD-PI/L whereas higher glucose concentrations lead to both lower specific and overall yields due to depletion of critical medium components in the production period. In fed-batch experiments at 30 degrees C it has been possible to obtain high specific SOD-PI yields even at high biomass concentrations by feeding glucose at a constant rate of 1.5 g/L/h for 40 h followed by a feeding of ethanol at 1.0 g/L/h for 24 h, thus giving an overall yield of 1200 mg/L. Decreasing the temperature from 30 to 26 degrees C leads to improved yields in batch as well as fed-batch experiments. The optimized fed-batch fermentation process which is suitable to be scaled up to the cubic meter level has been tested in 200-L fermentations resulting in yields of more than 1500 mg/L of the fusion protein which conveniently can be used as a precursor in the production of recombinant human proinsulin.

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

Abstract: 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.

Dynamic model development for a continuous culture of Saccharomyces cerevisiae

Abstract: The problem of dynamically modeling a chemostat is addressed. Using the results of continuous culture experiments for the growth of a strain of Saccharomyces cerevisiae on a glucose-limited medium, a general approach to developing dynamic models is discussed. The approach to develop and verify the model involves three different types of experiments: steady-state, dynamic step response, and feedback identification.

Competition for ethanol between sulfate-reducing and fermenting bacteria

Abstract: Competition for ethanol between the sulfate-reducing bacteria Desulfobulbus propionicus, Desulfotomaculum orientis, Desulfovibrio vulgaris Marburg, Desulfovibrio gigas, Desulfovibrio desulfuricans Essex and the fermenting bacteria Pelobacter propionicus and Acetobacterium carbinolicum were studied in batch culture. A number of these bacteria was also chosen for competition experiments under ethanol limitation in chemostat culture. The maximum growth rates determined by washout experiments were higher for the fermenting bacteria (μmax=0.096 resp. 0.335h−1) than for the sulfate-reducing bacteria (μmax≈0.03h−1). In contrast, the saturation concentrations for half maximum growth rates (Ks values) for ethanol were lower for the sulfate-reducing bacteria (Ks≈5 μM) than for the fermenting bacteria (Ks≥50 μM). In batch culture competition experiments the fermenting bacteria turned out to be the better competitors, degrading 51–80% of the ethanol added. In competition experiments with ethanollimited chemostat cultures the sulfate-reducing bacteria Desulfobulbus propionicus and Desulfovibrio vulgaris outcompeted Pelobacter propionicus at dilution rates below their maximum specific growth rates. At a high dilution rate, a fast growing population of Desulfobulbus propionicus originated and was enriched in the chemostat during the competition experiment.

Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153).

Abstract: Summary: The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HB101 have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0·3 h−1 than at 0·15 h−1. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0·15 h−1, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene. Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that in complex media the plasmid is maintained longer by E. coli HB101 at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.

Properties of Trichomonas vaginalis grown under chemostat controlled growth conditions.

Abstract: Trichomonas vaginalis isolates NYH 286 and IR 78 were grown in continuous flow culture conditions in a complex trypticase-yeast extract-maltose medium supplemented with heat-inactivated horse serum. Parasites could be stably maintained in the chemostat at high densities ranging from 1 x 10(6) to 1 x 10(7) organisms ml-1. Growth densities, acid production, and profiles of total versus secreted trichomonad proteins were characterised at different rates of growth and pH. Growth rate influenced the extent of parasite production of acid and the shedding of proteins into the medium but had no effect on overall parasite density. Lowering the pH from 6.0 to 5.0 resulted both in a decrease of cell density and acid production. At pH 4.5 isolate IR 78 but not NYH 286 was capable of growth and multiplication, showing the ability of some isolates to survive at the vaginal pH of healthy individuals. At this lower pH, however, isolate NYH 286 but not IR 78 synthesised new proteins which were detectable in stained gels. Also, inoculation of the chemostat with isolate NYH 286 comprising a mixture of fluorescent (positive, pos) and non-fluorescent (negative, neg) trichomonads as defined by monoclonal antibody reactivity to a surface immunogen resulted in a change in the parasite population to an almost homogeneous neg phenotype. These neg phenotype organisms switched back to pos phenotype after transfer to test tubes.

Chemostat studies of a mixed culture growing on phenolics.

Abstract: The growth of a mixed culture was studied in a two stage chemostat with growth limited by phenolics. The first stage provided a fully viable, adapted population that was fed at a constant rate to the second stage, where the concentration of phenolics could be varied. The population exhibited inhibition kinetics at high concentrations of phe nolics. The viability of the culture decreased with increasing dilution rate, or with increasing phenolic concentration. The nonviable population did not utilize significant amounts of substrate, but ignoring viability can lead to a significant underestimate of the growth rate. The mainte nance requirements were insignificant at noninhibitory levels of phenolics, but rose to 0.15 g/g*h at inhibitory levels, possibly due to the need to repair damaged cell membranes. Res. J. Water Pollut. Control Fed., 62, 684(1990).

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4.

Abstract: Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h−1) nitrate and nitrite reductase activities of 200 nmol · mg−1 protein · min−1 and 250 nmol · mg−1 protein · min−1 were measured, respectively. At a high growth rate (0.55 h−1) both enzyme activities were considerably lower (25 and 12 nmol mg−1 · protein · min−1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.

Mixotrophic and Autotrophic Growth of Thiobacillus acidophilus on Glucose and Thiosulfate

Abstract: Mixotrophic growth of the facultatively autotrophic acidophile Thiobacillus acidophilus on mixtures of glucose and thiosulfate or tetrathionate was studied in substrate-limited chemostat cultures. Growth yields in mixotrophic cultures were higher than the sum of the heterotrophic and autographic growth yields. Pulse experiments with thiosulfate indicated that tetrathionate is an intermediate during thiosulfate oxidation by cell suspensions of T. acidophilus. From mixotrophic growth studied, the energetic value of thiosulfate and tetrathionate redox equivalents was estimated to be 50% of that of redox equivalents derived from glucose oxidation. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) activities in cell extracts and rates of sulfur compound oxidation by cell suspensions increased with increasing thiosulfate/glucose ratios in the influent medium of the mixotrophic cultures. Significant RuBPCase and sulfur compound-oxidizing activities were detected in heterotrophically grown T. acidophilus. Polyhedral inclusion bodies (carboxysomes) could be observed at low frequencies in thin sections of cells grown in heterotrophic, glucose-limited chemostat cultures. Highest RuBPCase activities and carboxysome abundancy were observed in cells from autotrophic, CO{sub 2}-limited chemostat cultures. The maximum growth rate at which thiosulfate was still completely oxidized was increased when glucose was utilized simultaneously. This, together with the fact that even during heterotrophic growth the organism exhibited significant activities ofmore » enzymes involved in autotrophic metabolism, indicates that T. acidophilus is well adapted to a mixotrophic lifestyle. In this respect, T. acidophilus may have a competitive advantage over autotrophic acidophiles with respect to the sulfur compound oxidation in environments in which organic compounds are present.« less

A mutant of Mycoplasma mycoides subsp. mycoides lacking the H2O2‐producing enzyme l‐α‐glycerophosphate oxidase

Abstract: Cells of Mycoplasma mycoides subsp. mycoides grown without stirring or aeration in batch culture, and resuspended in a salts solution, oxidised a range of carbohydrates including glycerol. The rate of glycerol oxidation was not reduced when cells were passaged than 20 times in batch culture. However, in cells grown in stirred and aerated chemostat culture for 100 generations the ability to oxidise glycerol, but not other carbohydrates, was lost or greatly reduced. A mutant strain isolated from chemostat culture did not revert to glycerol utilisation even after several passages in batch culture. The growth rate and growth-yield of the mutant strain in batch culture were similar to those of the parent strain. The mutant possessed activity for glycerol kinase but had lost that for the hydrogen peroxide-producing enzyme-L-α-glycerophosphate oxidase. The selection pressure in favour of the mutant starin in chemostat culture may be a decreased production of hydrogen peroxide.

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions.

Abstract: Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.