Der substratlimitierte pH-Auxostat – Eine neue Methode zur kontinuierlichen Kultur

A method for continuous cultivation of microorganisms is demonstrated, the substrate limited pH-auxostat. The limiting substrate only is added with constant velocity. In this culture the cells grow with high utilization of the limiting substrate and with the highest specific growth rate possible at the given conditions. Yield coefficients and dilution rates of stable K⁺-limited steady states in yeast cultures with different pH-values and biomass concentrations were measured.     

The coupling between catabolism and anabolism of Methanobacterium thermoautotrophicum in H2- and iron-limited continuous cultures

The aim of the present work was to investigate whether uncoupling of catabolism from anabolism, which was often observed in heterotrophic microorganisms under energy-sufficient growth conditions, also occurs in the autotrophic bacterium Methanobacterium thermoautotrophicum. For this purpose, M. thermoautotrophicum was cultivated in continuous cultures that were limited by the trace element iron. The influences of both dilution rate and iron supply rate on the coupling between anabolism and catabolism were investigated. As compared to continuous cultures of M. thermoautotrophicum limited by the energy substrate H₂, a 5-fold decrease in the biomass concentration and a 3-fold decrease in H₂, CO₂, and CH₄ conversion rates were observed in iron-limited cultures. However, the specific substrate and product conversion rates increased as compared to the values determined in energy-limited cultures. Thus, iron limitation provoked an uncoupling of catabolism from anabolism. At a dilution rate of 0.096 h⁻¹ and at an iron concentration of 17 μM in the feed, the specific H₂ consumption rate was 100% higher than the rate determined under H₂-limiting conditions, whereas at a dilution rate of 0.168 h⁻¹, the values differed only by 5%. Uncoupling of catabolism from anabolism also increased dramatically when the iron supply rate was lowered but the dilution rate was kept constant. Thus, the extent of uncoupling is a function of both the dilution rate and the iron supply rate. It was found that the specific consumption rate of H₂ increased in parallel with the partial pressure of H₂ in the culture medium. This suggested that the catabolic activity of M. thermoautotrophicum was not stringently controlled at the enzymatic level and can be considerably stimulated by the excess of H₂ in the medium. Hypotheses as to the fate of the excess energy derived from uncoupled catabolism are discussed, but the physiological reason for the partial uncoupling between catabolism and anabolism remains yet to be clarified.     

Metabolic regulation in bacterial continuous cultures: II

The transient behavior of a continuous culture of Klebsiella pneumoniae with mixed feed of glucose and xylose arising from step-up and step-down in dilution rates and from a feed-switching experiment is presented. he organism gradually switches from simultaneous utilization of the substrates at low growth rates to preferred utilization of the faster substrate (i.e, supporting a higher growth rate) at high dilution rates. The metabolic lags following a step increase in dilution rate and a significant accumulation of the slower substrate during the transient period result from the effects of metabolic regulation. The cybernetic modeling approach that successfully described the foregoing situations with single-substrate feeds is employed to describe mixed substrate behavior. The parameters in the mixed-substrate (glucose and xylose) model are the same as those in the single-substrate models with the singular exception of the rate constant for the xylose growth enzyme synthesis. The reason for this discrepancy is discussed in detail. It appears that the constitutive rate of enzyme synthesis for growth on a given substrate may be related to the past history of the organism in regard to whether or not the organism has been exposed to the particular substrate. Thus, the results further demonstrate the ability of the framework to effectively describe metabolic regulation in batch, fedbatch, and continuous microbial cultures.     

Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cells

The kinetic behavior of a nonproducing hybridoma clone AFP-27-NP was investigated in continuous culture under glucose-limited conditions. A total of more than 21, 000 h of cultures were operated at dilution rates ranging from 0.01 to 0.06 h(-1). The viable cell concentrations, dead cell concentrations, and cell volumes all varied with the dilution rate. A steady-state model was developed based on the biomass concentration and the glucose concentration. The specific growth rate as a function of glucose concentration is described by a model similar to the Monod model with a threshold glucose concentration and a minimum specific growth rate incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. A death rate is included in the model which is described by an inverted Monod-type function of glucose concentration. The yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper region of specific growth rates. No maintenance term for glucose consumption was needed; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepted the specific growth rate axis at a value close to the minimum growth rate. The values for the model parameters were determined from regression analysis of the steady-state data. The model predictions and experimental results fit very well.     

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.     

Glucose uptake rates of single E. coli cells grown in glucose-limited chemostat cultures

To evaluate the extent to which single-cell glucose uptake rates determine the overall specific growth rate of a culture, dilute chemostat cultures of Escherichia coli BL21 were grown in defined medium under glucose limitation. The glucose uptake dynamics of the cell population was examined at the single-cell level using the fluorescent glucose analog, 2-NBDG. Between dilution rates of 0.12 h(-1) and 0.40 h(-1), mean cellular protein content and steady-state, extracellular glucose concentrations increased with increasing dilution rate. However, the distribution of 2-NBDG uptake rates in the population remained constant over the range of dilution rates studied. This indicates that the growth of cells in continuous culture is not limited by the maximum rate of uptake of glucose but by the availability of glucose for transport. The work represents an example of how quantitative flow cytometry can be applied to gain detailed insight into microbial growth physiology.     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors

Plant cell suspension cultures of Catharanthus roseus and Nicotiana tabacum were grown in stirred tank bioreactors operated in batch and continuous mode. The stoichiometry of growth of both species in steady-state glucose limited chemostats was studied at a range of different dilution rates. A linear relation was applied to describe specific glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate. Specific respiration deviated greatly from the linear relation. An unstructured mathematical model, based on the observed stoichiometry in the glucose limited chemostats, was applied to describe the growth in batch culture. From a comparison between the observed growth pattern in batch fermentors and computer simulations it appeared that the stoichiometry of growth of the C. roseus culture was different under steady-state and dynamic conditions. It was concluded that a mathematical model for the growth of suspension culture plant cells in which the biomass is considered to be a single compound with an average chemical composition is of limited value because large changes in the conmposition of the biomass may occur. (c) 1992 John Wiley & Sons, Inc.     

On-line growth measurements in bioreactors by titrating metabolic proton exchange

Recording the amount of titrant required to maintain constant pH in a bioreactor where cell metabolism causes acidity changes allows on-line determinations of growth kinetics in computer-controlled batch cultures. A system for making such measurements is described and its performance is investigated. Transient bicarbonate accumulation occurs if the culture produces CO₂ at high pH values and low gas transfer rates. We have developed a mathematical model for the titrant requirement as a function of the cell growth rate, the gas transfer properties of the bioreactor and the culture pH. According to this model, bicarbonate accumulation affects the stoichiometry between titrant and biomass but does not prevent determination of growth rate constants. These predictions are confirmed using model experiments and measurements during batch growth of microbial cultures.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Inhibition Kinetics of Phenol Degradation by Pseudomonas aeruginosa from Continuous Culture and Washout Data

Steady states of a continuous culture with an inhibitory substrate were used to estimate kinetic parameters under substrate limitation (chemostat operation). Pure cultures of an indigenous Pseudomonas aeruginosa were grown in continuous culture on phenol, the sole source of carbon and energy, at dilution rates of 0.010 to 0.20 h^{- 1}. Using different dilution rates, several steady states were investigated and the specific phenol consumption rates were calculated. In addition, phenol degradation was investigated by increasing the dilution rate above the critical dilution rate (washout cultivation). The results showed that the specific phenol consumption rate increased with increased dilution rate at steady state and that the degradation by Pseudomonas aeruginosa can be described by simple substrate inhibition kinetics under substrate limitation but cannot be described by simple substrate inhibition kinetics under washout cultivation. Fitting of the steady-state data from continuous cultivation to various inhibition models resulted in the best fit for the Yano and Koga kinetic inhibition model. The r_{s max} value of 0.278 mg/mg/h obtained from the Yano and Koga equation was comparable to the experimentally calculated r_{s max} value of 0.283 mg/mg/h obtained under washout cultivation.     

Transient behavior of a continuous stirred tank biological reactor utilizing phenol as an inhibitory substrate

Transient experiments were conducted on a Pseudomomas utilizing phenol in a continuous culture by disturbing the influent substrate concentration and dilution rate. Two stable steady states existed for some ranges of the parameters. Highly damped oscillations were observed in approaching a new high conversion steady state or in returning to a new high conversion steady state following a small disturbance. When a large disturbance was applied there was a smooth (overdamped) approach to a new low conversion steady state. The observed oscillatory behavior for small disturbances was predicted by a modified Powell-Ierusalemskii bottleneck model, but could not be predicted by a Monod-Haldane model; neither model was accurate for predicting the effect of large disturbances. A constant wall growth factor was used to account for microbial film activity, and the existence of two stable states was directly due to the presence of the film.     

Effect of oxygen supply on growth ofKlebsiella aerogenes in a multistage tower fermentor

The effect of the rate of oxygen supply on biomass growth, consumption of carbon source formation of metabolic by-products, biomass yeilds referred to C-source and oxygen, respiration rate and the respiratory quotient was studied in a multistage tower fermentor with an interstage backflow, i.e. with a continuous reinoculation of the preceding stages. Experiments were done with Klebsiella aerogenes CCM 2318 in a synthetic glucose medium with constant glucose concentration in the feed, at pH 7.0. temperature 30 degrees C, and dilution rates 0.6 and 0.178 h-1 (referred to one stage). Different behavior of the culture was found at different dilution rates both with oxygen and under oxygen limitation. As compared with the chemostat system, the regime with an interstage backflow exhibited differences in respiration rate and CO2 formation; this attests to a considerably different physiological state of the cells.     

Evaluation of yield and maintenance coefficients, expressed in carbon units, for Pseudomonas fluorescens and P. aeruginosa.

The maximum cell yield (YMc - g biomass carbon per gram substrate carbon) and the rate of maintenance metabolism (mc - g substrate carbon/g biomass carbon per hour) have been determined for substrate limited continuous cultures of Pseudomonas fluorescens and P. aeruginosa. The metabolism of the organic substrates was monitored by measuring the COD-removal1) rates at different dilution rates. The advantages of expressing yield and maintenance coefficients in carbon units is discussed.     

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.     

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Hopf bifurcation for a family of two-state microbial growth models

We describe a family of two-state microbial growth models, in which growth and maintenance are assigned to two different cell states. The way of splitting periodic solutions for low dilution rates of a continuous fermentation is shown. The existence of these periodie solutions is mainly influenced by the properties of substrate consumption, which the maintenance rate in the second cell state amounts to.     

Gene Regulation in Continuous Cultures: A Unified Theory for Bacteria and Yeasts

During batch growth on mixtures of two growth-limiting substrates, microbes consume the substrates either sequentially (diauxie) or simultaneously. The ubiquity of these growth patterns suggests that they may be driven by a universal mechanism common to all microbial species. Recently, we showed that a minimal model accounting only for enzyme induction and dilution, the two processes that occur in all microbes, explains the phenotypes observed in batch cultures of various wild-type and mutant/recombinant cells (Narang and Pilyugin in J. Theor. Biol. 244:326–348, 2007). Here, we examine the extension of the minimal model to continuous cultures. We show that: (1) Several enzymatic trends, attributed entirely to cross-regulatory mechanisms, such as catabolite repression and inducer exclusion, can be quantitatively explained by enzyme dilution. (2) The bifurcation diagram of the minimal model for continuous cultures, which classifies the substrate consumption pattern at any given dilution rate and feed concentrations, provides a precise explanation for the empirically observed correlations between the growth patterns in batch and continuous cultures. (3) Numerical simulations of the model are in excellent agreement with the data. The model captures the variation of the steady state substrate concentrations, cell densities, and enzyme levels during the single- and mixed-substrate growth of bacteria and yeasts at various dilution rates and feed concentrations. This variation is well approximated by simple analytical expressions that furnish deep physical insights. (4) Since the minimal model describes the behavior of the cells in the absence of cross-regulatory mechanisms, it provides a rigorous framework for quantifying the effect of these mechanisms. We illustrate this by analyzing several data sets from the literature.