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1.
Two novel, nitrogen-limited and oxygen-limited, perturbed transient experiments were performed to examine the presence of active transport of methanol and study the effect on methanol uptake by a methanol-utilizing bacterium, L3, in a batch bioreactor. Transient limitations of both ammonium ions and O(2) in batch cultures were found to cause methanol leakages out of the cells, suggesting the presence of an active transport of methanol in L3. Such experimental results were used to indirectly estimate the intracellular levels of methanol during a batch growth of L3. The results of our analysis indicate that the intracellular methanol level is high and show an increasing trend during the unbalanced phase, but falls to a constant low level in the balanced phase of a typical batch growth(8) of L3. A simple modelling analysis suggests that a four-parameter "pump-and-extrusion" model could be used to adequately describe the transport of methanol between the extra cellular and intracellular phases of batch cultures of L3.  相似文献   

2.
Bacterial cells have three phases of growth which are characterized respectively by: (1) balanced growth with a high yield of biomass; (2) balanced growth with lower biomass yield; and (3) unbalanced growth with lowest biomass yield. Phases 2 and 3 are associated with elevated concentrations of the regulatory nucleotides centered on guanosine-5′-diphosphate-3′-diphosphate. Maintenance of the correct growth phase is important in optimizing industrial product formation by bacterial populations.  相似文献   

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5.
It is crucial to the reproducibility of results and their proper interpretation that the conditions under which experiments are carried out be defined with rigour and consistency, in this review we attempt to clarify the differences and interrelationships among steady, balanced and exponential states of culture growth. Basic thermodynamic concepts are used to introduce the idea of steady-state growth in open, biological systems. The classical, sometimes conflicting, definitions of steady-state and balanced growth are presented, and a consistent terminology is proposed. The conditions under which a culture in balanced growth is also in exponential growth and in steady-state growth are indicated. It is pointed out that steady-state growth always implies both balanced and exponential growth, and examples in which the converse does not hold are described. More complex situations are then characterized and the terminology extended accordingly. This leads to the notion of normal growth and growth that can be synchronous or otherwise unbalanced but still reproducible, and to the condition of approximate steady state manifested by growth in batch culture and by asymmetrically dividing cells, which is analysed in some detail.  相似文献   

6.
Butyribacterium methylotrophicum is an anaerobic bacterium that can convert methanol to butyrate. This ability to produce longer-chain carbon compounds from C(1) substrates could be of commercial significance. The fermentation rates and product formation depend on the methanol/bicarbonate ratios during fermentation. The kinetics of batch fermentation fit the Luedeking-Piret model with growth and maintenance associated product formation. Butyrate yield of 0.256 mol/mol methanol (ca. 85% of theoretical yield) has been obtained in batch fermentation.  相似文献   

7.
In biological oceanography, it has been widely accepted that the maximum quantum yield of photosynthesis is influenced by nutrient stress. A closely related parameter, the maximum quantum yield for stable charge separation of PSII, (φ PSII )m, can be estimated by measuring the increase in fluorescence yield from dark-adapted minimal fluorescence (Fo) to maximal fluorescence (Fm) associated with the closing of photosynthetic reaction centers with saturating light or with a photosynthetic inhibitor such as 3′-(3,4-dichlorophenyl)-1′,1′-dimethyl urea (DCMU). The ratio Fv/Fm (= (Fm− Fo)/Fm) is thus used as a diagnostic of nutrient stress. Published results indicate that Fv/Fm is depressed for nutrient-stressed phytoplankton, both during nutrient starvation (unbalanced growth) and acclimated nutrient limitation (steady-state or balanced growth). In contrast to published results, fluorescence measurements from our laboratory indicate that Fv/Fm is high and insensitive to nutrient limitation for cultures in steady state under a wide range of relative growth rates and irradiance levels. This discrepancy between results could be attributed to differences in measurement systems or to differences in growth conditions. To resolve the uncertainty about Fv/Fm as a diagnostic of nutrient stress, we grew the neritic diatom Thalassiosira pseudonana (Hustedt) Hasle et Heimdal under nutrient-replete and nutrient-stressed conditions, using replicate semicontinuous, batch, and continuous cultures. Fv/Fm was determined using a conventional fluorometer and DCMU and with a pulse amplitude modulated (PAM) fluorometer. Reduction of excitation irradiance in the conventional fluorometer eliminated overestimation of Fo in the DCMU methodology for cultures grown at lower light levels, and for a large range of growth conditions there was a strong correlation between the measurements of Fv/Fm with DCMU and PAM (r2 = 0.77, n = 460). Consistent with the literature, nutrient-replete cultures showed consistently high Fv/Fm (∼0.65), independent of growth irradiance. Under nutrient-starved (batch culture and perturbed steady state) conditions, Fv/Fm was significantly correlated to time without the limiting nutrient and to nutrient-limited growth rate before starvation. In contrast to published results, our continuous culture experiments showed that Fv/Fm was not a good measure of nutrient limitation under balanced growth conditions and remained constant (∼0.65) and independent of nutrient-limited growth rate under different irradiance levels. Because variable fluorescence can only be used as a diagnostic for nutrient-starved unbalanced growth conditions, a robust measure of nutrient stressed oceanic waters is still required.  相似文献   

8.
A combined analysis of growth and metabolite composition was performed in primary roots of Zea mays L. (Var. Alexander). The seedlings were hydroponically cultivated either in pure water or in complete nutrient solution. The overall root growth performance was similar in both treatments. Yet, digital image sequence processing methods resolved, that growth distribution and oscillatory movements within the growth zone depended strongly on external nutrient availability. Metabolite concentration profiles were similar in both treatments for most investigated metabolites, indicating a thorough mobilization of nutrient resources from the seed, but concentrations of glutamine, glutamic acid, NO3, NH4+, malate and citrate showed pronounced differences between treatments. No diurnal variations in metabolite concentrations were found. Deposition rate profiles were in general more similar to relative elemental growth rate profiles than concentration profiles and were not affected by the treatment. Major ions were deposited maximally in front of the centre of growth activity, while greatest hexose deposition was found behind that. Relative to their abundance in the root growth zone, net rates of transfer from mature tissue were highest for sucrose, glutamic acid and aspartic acid, whereas glucose, fructose and most amino acids inversely showed high net rates of transfer out of the root growth zone, indicating a high catabolic rate for those substances there. NO3, but not other nutrients, was transferred to a great extent from the root growth zone to the mature tissue in nutrient solution. Overall, the results show, that a careful analysis of growth dynamics allows quantifying and interpreting a number of important flux parameters in the growing organ and that the performance of the primary root does not depend strongly on external nutrient availability.  相似文献   

9.
Attaining metabolic and isotopic balanced growth is one critical condition for physiological studies using isotope-labeled tracers, but is very difficult to obtain in batch culture due to the extensive metabolite exchange with the surrounding medium and related physiological changes. In the present study, we investigated metabolic and isotopic behavior of CHO cells in differently designed media. We observed that the assumption of balanced cell growth cannot be justified in batch culture of CHO cells directly using conventional, commercially available media. By systematically redesigning media composition and characterizing metabolic steady state based on mass balances and measurement of labeling dynamics, we achieved balanced cell growth for the main cellular substrates in CHO cells. This was done in a step-by-step analysis of growth and primary metabolism of CHO cells with the use of [U-13C]glucose feeding and adjusting concentrations of amino acids in the growth medium. The optimized media obtained at the end of the study provide balanced growth and isotopic steady state or at least asymptotic steady state. As a result, we established a platform to conduct isotope-based physiological studies of mammalian systems more reliably and therefore well suited for later use in metabolic profiling of mammalian systems such as 13C-labeled metabolic flux analysis.  相似文献   

10.
Summary The most direct approach to enhancing the volumetric yield of secondary metabolites in plant tissue cultures is to operate the culture under high cell density. In this study, a cell suspension ofAnchusa officinalis was cultivated using a semi-continuous perfusion technique, i.e. batch cultivation with intermittent medium exchange. Using a perfusion medium containing sucrose concentration which was two times that in the normal growth medium, the final cell density and the final product concentration were increased by more than 2-fold compared with a batch culture without medium exchange. The high cell density obtained from the semi-continuous perfusion culture can be explained by the prevention of nutrient depletion, removal of toxic by-products, as well as the control of cell size by virtue of the high sugar medium osmolarity.  相似文献   

11.
The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.  相似文献   

12.
A miniaturized reactor system with on‐line measurement of respiration rates by membrane inlet mass spectrometry was applied for the on‐line metabolic flux analysis at different phases of a 1.2 L batch culture of lysine‐producing Corynebacterium glutamicum. For this purpose, cells taken from the batch culture were transferred into the 12 mL mini reactor, and incubated for 15 min with [1‐18O]glucose. Quantification of oxygen uptake rate and CO2 mass isotopomer production rates in combination with a simple metabolic model allowed the estimation of the flux partitioning ratio between the pentose phosphate pathway and glycolysis during the process. The relative flux into the pentose pathway increased during growth, and reached maxima at 11 and 17 h cultivation time coinciding with maxima of the differential lysine yield. The developed system is a promising tool for determination of metabolic flux dynamics in industrially relevant batch and fed‐batch cultures.  相似文献   

13.
Production of bio-pharmaceuticals in cell culture, such as mammalian cells, is challenging. Mathematical models can provide support to the analysis, optimization, and the operation of production processes. In particular, unstructured models are suited for these purposes, since they can be tailored to particular process conditions. To this end, growth phases and the most relevant factors influencing cell growth and product formation have to be identified. Due to noisy and erroneous experimental data, unknown kinetic parameters, and the large number of combinations of influencing factors, currently there are only limited structured approaches to tackle these issues. We outline a structured set-based approach to identify different growth phases and the factors influencing cell growth and metabolism. To this end, measurement uncertainties are taken explicitly into account to bound the time-dependent specific growth rate based on the observed increase of the cell concentration. Based on the bounds on the specific growth rate, we can identify qualitatively different growth phases and (in-)validate hypotheses on the factors influencing cell growth and metabolism. We apply the approach to a mammalian suspension cell line (AGE1.HN). We show that growth in batch culture can be divided into two main growth phases. The initial phase is characterized by exponential growth dynamics, which can be described consistently by a relatively simple unstructured and segregated model. The subsequent phase is characterized by a decrease in the specific growth rate, which, as shown, results from substrate limitation and the pH of the medium. An extended model is provided which describes the observed dynamics of cell growth and main metabolites, and the corresponding kinetic parameters as well as their confidence intervals are estimated. The study is complemented by an uncertainty and outlier analysis. Overall, we demonstrate utility of set-based methods for analyzing cell growth and metabolism under conditions of uncertainty.  相似文献   

14.
A simple mathematical model describing the cell cycle dependency of rice alpha-amylase production by a recombinant yeast was constructed to investigate the efficiency of cell cycle population control. First, the effects of the glucose concentration and cultivation temperature on the specific growth rate, the specific production rate of rice alpha-amylase, and the distribution of the cell cycle population were studied under balanced growth conditions. On the basis of the results, parameter values for the mathematical model were then estimated. The proposed model was shown to be applicable for unbalanced as well as balanced growth phases. The optimal control strategy in respect of temperature and glucose concentration for maximum rice alpha-amylase production, taking into account the cell cycle population, was determined and the result was compared with that obtained by a simple mathematical model in which cell cycle distribution was not considered. Finally, the effect of the initial population of each cell cycle phase on the final amount of the product under optimal operational conditions was investigated. The simulation and experimental data coincided well with each other, and the model was used to optimize the control strategy for maximum alpha-amylase production.  相似文献   

15.
Growth characteristics of batch and continuous cultures of the pink facultative methylotrophMethylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C1-metabolism were measured in a chemostat culture at different dilution rates.  相似文献   

16.
A methodology is developed that determines age-specific transition rates between cell cycle phases during balanced growth by utilizing age-structured population balance equations. Age-distributed models are the simplest way to account for varied behavior of individual cells. However, this simplicity is offset by difficulties in making observations of age distributions, so age-distributed models are difficult to fit to experimental data. Herein, the proposed methodology is implemented to identify an age-structured model for human leukemia cells (Jurkat) based only on measurements of the total number density after the addition of bromodeoxyuridine partitions the total cell population into two subpopulations. Each of the subpopulations will temporarily undergo a period of unbalanced growth, which provides sufficient information to extract age-dependent transition rates, while the total cell population remains in balanced growth. The stipulation of initial balanced growth permits the derivation of age densities based on only age-dependent transition rates. In fitting the experimental data, a flexible transition rate representation, utilizing a series of cubic spline nodes, finds a bimodal G(0)/G(1) transition age probability distribution best fits the experimental data. This resolution may be unnecessary as convex combinations of more restricted transition rates derived from normalized Gaussian, lognormal, or skewed lognormal transition-age probability distributions corroborate the spline predictions, but require fewer parameters. The fit of data with a single log normal distribution is somewhat inferior suggesting the bimodal result as more likely. Regardless of the choice of basis functions, this methodology can identify age distributions, age-specific transition rates, and transition-age distributions during balanced growth conditions.  相似文献   

17.
The behavior of Enterobacter aerogenes during growth in chemostats limited by single and double nutrient restrictions was examined. On the assumption that different essential nutrients act to limit growth in different ways, we selected pairs of nutrients likely to affect different aspects of metabolism. Results show that macromolecular cell composition can be controlled by using more than one nutrient restriction. The polysaccharide content of the cells is readily manipulated by the ratio of carbon to nitrogen in the inlet nutrients. Also, at low dilution rates, ratios of protein to ribonucleic acid are dependent on the ratio of phosphate to nitrogen in the input nutrients. An examination of both acetic acid and metabolite production (as measured by ultraviolet absorbance of culture filtrates) showed that accumulation of these products was dependent on both dilution rate and type of nutrient limitation(s). These results were examined in terms of the problems of translation of batch to continuous culture processes and the use of selected nutrient limitations to control noncellular product formation.  相似文献   

18.
The development of serum- and protein-free Chinese hamster ovary (CHO) cell cultures is a high priority for the production of biopharmaceuticals. Protein-free competent CHO cells lines have been previously constructed by two different methods-metabolic engineering with cell-cycle regulatory proteins and long-term selective adaptation. Apoptosis was present in both cell lines during protein-free, static-batch culture as a result of nutrient deprivation, and glucose deprivation alone was a potent inducer of apoptosis compared to the depletion of other nutrients such as amino acids. By adding back serum to the cultures during batch growth or nutrient deprivation, it was shown that unidentified survival factors in serum can greatly reduce apoptosis in protein-competent cell lines in all phases of the culture. Both observations contrast to previous reports for hybridoma cells, in which amino acids were the key determinants of apoptosis and serum had no additional antiapoptotic effect. Serum's protective effect against CHO cell death in batch culture was multifaceted and complex: (1) 10% FBS increased cell viability to >99% during exponential growth from roughly 75-90%, (2) 5-10% fetal bovine serum (FBS) reduced specific glucose consumption rates in both cell lines by 40%, thereby delaying the onset of apoptosis caused by glucose deprivation, and (3) 5% FBS reduced the specific cell death rate by 65% during a 3-d lactate-consumption phase characterized by substantial abortive proliferation, in which the cells both proliferated and died at a constant rate. The benefit of serum on cell production over the various phases of batch growth was combined into a single parameter by integrating the viable cell concentration vs. time profile (termed here as cumulative volumetric viable cell-time, VCTvol). Despite the ability of both cell lines to grow indefinitely without any exogenous growth factors, the addition of serum resulted in a 2. 3-fold increase in the VCTvol. Thus, it is clear that there is much room for improvement of protein-free CHO cell lines despite their adequate growth competence, and new strategies different from those successfully used for hybridomas may be necessary to combat CHO cell apoptosis.  相似文献   

19.
Mixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time-consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly-control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs-controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical-specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time-dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment.  相似文献   

20.
Acetone-butanol-ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12- and 20-in.-long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18-6.43 to 4.50-4.90 in the first inch of the reactor. The pH dropped further to 4.36-4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and nutrient supply only to the inlet of the reactor.  相似文献   

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