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  • Articles  (66)
  • continuous culture  (66)
  • Process Engineering, Biotechnology, Nutrition Technology  (66)
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 115-117 
    ISSN: 1573-0972
    Keywords: Candida utilis ; pineapple cannery effluent ; continuous culture ; steady-state
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Candida utilis was grown on a pineapple cannery effluent in a chemostat at dilution rates ranging between 0.05 and 0.65 h−1 to establish optimal conditions for biomass production and chemical oxygen demand (COD) reduction. Sucrose, fructose and glucose were the main sugars in the effluent. Maximum value for cell yield coefficient and productivity were (0.686, gx/gs) and (2.96, gx/l/h) at a dilution rate of 0.425 and 0.475 h−1, respectively, while maximum COD reduction (98%) was attained at a dilution rate of 0.1 h−1. The maintenance coefficient attained a value of (0.093, gs/gx/h). An increase in dilution rate produced a higher protein content of the biomass.
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  • 2
    Electronic Resource
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    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 235-238 
    ISSN: 1573-0972
    Keywords: Arthrospira platensis ; bioenergetic yield ; continuous culture ; irradiance ; specific growth rate ; mixotrophy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Reassessement of bioenergetic growth yield of Arthrospira platensis was performed by using continuous culture under both autotrophic and mixotrophic conditions. Continuous culture was carried out at dilution rates of 0.017, 0.023 and 0.030 h−1. Under these dilution rates bioenergetic yields ranged between 4.45–6.03 × 10−3 g biomass kJ−1 and between 5.42–7.46 × 10−3 g biomass kJ−1, under autotrophic and mixotrophic conditions respectively. A maximum bioenergetic yield of 8.1 × 10−3 g biomass kJ−1 using an autotrophic culture can be calculated. Pigment accumulation (chlorophyll a and carotenoids) may be related to light irradiance, reaching a maximum pigment concentration under light saturation irradiance. Phycocyanin concentration increased during light limitation.
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  • 3
    ISSN: 1573-0972
    Keywords: Acetic acid production ; carbon metabolism ; continuous culture ; Escherichia coli ; metabolic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO2 production, and in oxygen consumption.
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  • 4
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    Cytotechnology 29 (1999), S. 71-84 
    ISSN: 1573-0778
    Keywords: bioreactor ; continuous culture ; hybridoma cells ; hyperosmolality ; monoclonal antibody production ; non-producing subpopulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase.
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  • 5
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO ; continuous culture ; flow cytometry ; perfusion culture ; spin-filter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures.
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  • 6
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    Cytotechnology 31 (1999), S. 243-254 
    ISSN: 1573-0778
    Keywords: continuous culture ; growth inhibition ; osmolality ; perfusion culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Continuous culture is frequently used in the cultivation of mammalian cells for the manufacturing of recombinant protein pharmaceuticals. In such operations a large volume of medium is turned over each day, especially in the case where cell recycle, or perfusion cultivation, is practiced. In principle, the volumetric throughput of medium can be reduced by using a more concentrated feed while maintaining the same nutrient provision rate. Overall, the medium components are divided into two categories: ‘consumable nutrients' and ‘unconsumable inorganic bulk salts’. In such fortified medium, the concentrations of consumable nutrients, but not bulk salts, are increased. With a stoichiometrically-balanced medium, the large amount of nutrients fed into the culture is largely consumed by cells to give rise to residual concentrations of these nutrients in their optimal range. However, unless care is taken to initiate the continuous culture, overshoot of nutrients may occur during the transient period. The high nutrient concentration during overshoot may be inhibitory by itself, or the resulting high osmolality may retard the growth. Using a mathematical model that incorporates the growth inhibitory effect of high osmolality we demonstrate such a potentially catastrophic effect of nutrient and osmolality overshoot by simulation. To avoid overshoot a controlled nutrient feeding scheme should be devised at the initiation of continuous culture.
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  • 7
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 62-70 
    ISSN: 0006-3592
    Keywords: plasmid stability ; recombinant microorganism ; continuous culture ; Pseudomonas sp. B13 FR1 pFRC20P ; degradation of aromatic compounds ; chlorobenzoate ; methylbenzoate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 62-70, 1998.
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  • 8
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    Biotechnology and Bioengineering 59 (1998), S. 99-107 
    ISSN: 0006-3592
    Keywords: Monod kinetics ; mixed substrate growth ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the μmax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:99-107, 1998.
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  • 9
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 251-261 
    ISSN: 0006-3592
    Keywords: continuous culture ; metabolic overflow ; multiplicity ; stability analysis ; dynamics ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 251-261, 1998.
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  • 10
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    Biotechnology and Bioengineering 57 (1998), S. 287-296 
    ISSN: 0006-3592
    Keywords: expression ; plasmid ; stability ; TCE ; continuous culture ; activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min · mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 287-296, 1998.
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  • 11
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    Biotechnology and Bioengineering 58 (1998), S. 617-624 
    ISSN: 0006-3592
    Keywords: thermoacidophile ; chemolithotroph ; heat shock ; chemical stress ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biooxidation capacity of an extremely thermoacidophilic archaeon Metallosphaera sedula (DSMZ 5348) was examined under bioenergetic challenges imparted by thermal or chemical stress in regard to its potential use in microbial bioleaching processes. Within the normal growth temperature range of M. sedula (70-79°C) at pH 2.0, upward temperature shifts resulted in bioleaching rates that followed an Arrhenius-like dependence. When the cells were subjected to supraoptimal temperatures through gradual thermal acclimation at 81°C (Han et al., 1997), cell densities were reduced but 3 to 5 times faster specific leaching rates (Fe3+ released from iron pyrite/cell/h) could be achieved by the stressed cells compared to cells at 79°C and 73°C, respectively. The respiration capacity of M. sedula growing at 74°C was challenged by poisoning the cells with uncouplers to generate chemical stress. When the protonophore 2,4-dinitrophenol (5-10 μM) was added to a growing culture of M. sedula on iron pyrite, there was little effect on specific leaching rates compared to a culture with no protonophore at 74°C; 25 μM levels proved to be toxic to M. sedula. However, a significant stimulation in specific rate was observed when the cells were subjected to 1 μM nigericin (+135%) and 2 μM (+63%); 5 μM levels of the ionophore completely arrested cell growth. The ionophore effect was further investigated in continuous culture growing on ferrous sulfate at 74°C. When 1 μM nigericin was added as a pulse to a continuous culture, a 30% increase in specific iron oxidation rate was observed for short intervals, indicating a potential positive impact on leaching when periodic chemical stress is applied. This study suggests that biooxidation rates can be increased by strategic exposure of extreme thermoacidophiles to chemical or thermal stress, and this approach should be considered for improving process performance. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 617-624, 1998.
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  • 12
    ISSN: 1573-0778
    Keywords: adenovirus major late promoter ; β-galactosidase ; Chinese hamster ovary cells ; continuous culture ; G1 phase expression ; inverse-growth associated production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production.
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  • 13
    ISSN: 1573-0778
    Keywords: antibody production ; carbon dioxide ; cell metabolism ; continuous culture ; inhibition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract CO2 partial pressure (pCO2) in industrial cell culture reactors may reach 150–200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. Due to equilibrium with bicarbonate, increased pCO2 at constant pH results in a proportional increase in osmolality. Hybridoma AB2-143.2 cell growth rate decreased with increasing pCO2 in well-plate culture, with a 45% decrease at 195 mm Hg with partial osmolality compensation (to 361 mOsm kg- 1). Inhibition was more extensive without osmolality compensation, with a 63% decrease in growth rate at 195 mm Hg and 415 mOsm kg-1. Also, the hybridoma death rate increased with increasing pCO2, with 31- and 64-fold increases at 250 mm Hg pCO2 for 401 and 469 mOsm kg- 1, respectively. The specific glucose consumption and lactate production rates were 40–50% lower at 140 mm Hg pCO2. However, there was little further inhibition of glycolysis at higher pCO2. The specific antibody production rate was not significantly affected by pCO2 or osmolality within the range tested. Hybridomas were also exposed to elevated pCO2 in continuous culture. The viable cell density decreased by 25–40% at 140 mm Hg. In contrast to the well-plate cultures, the death rate was lower at the new steady state at 140 mm Hg. This was probably due to higher residual nutrient and lower byproduct levels at the lower cell density (at the same dilution rate), and was associated with increased cell-specific glucose and oxygen uptake. Thus, the apparent effects of pCO2 may vary with the culture system.
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  • 14
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    World journal of microbiology and biotechnology 14 (1997), S. 113-118 
    ISSN: 1573-0972
    Keywords: Aspergillus ; continuous culture ; glucoamylase ; growth ; fungi ; nitrogen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Maltose-limited continuous culture of Aspergillus niger was carried out with potassium nitrate to investigate growth and glucoamylase formation characteristics. Glucoamylase production was dependent on the specific growth rate. The maximal amount of glucoamylase (units/l and U/g dry weight) was obtained at μ=0.08h−1, and the maximum specific rate of production (units/g/dry weight per hour) was at μ=0.2h−1. The maintenance coefficients (ms and mATP) were higher than for some other fungi. Maximal growth yields on substrate, oxygen and ATP (Yxsm, YxO2m and Yxam) were very efficient (high) and the value of Yxam, which cannot exceed the theoretical maximal value, is obtained when a P/O ratio of 1:1 is assumed. This indicates that biomass formation is energetically inexpensive and most of the expended energy has to be invested in the process of glucoamylase excretion.
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  • 15
    ISSN: 0006-3592
    Keywords: Bacillus stearothermophilus ; continuous culture ; plasmid stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The optimal culture conditions for Bacillus stearothermophilus NUB3621 (BGSC 9A5) in chemostat were studied. The results obtained showed that the optimal culture conditions in terms of biomass concentration and maximum growth rate were 65°C, pH 6.8 to 7.2. Dissolved oxygen became growth limiting at pO2 levels below 10%. Furthermore, this strain was transformed with three new hybrid vectors (pPAM2, pPCH2, or pPLY2) constructed by cloning in pRP9, a plasmid based on the thermophilic replicon, pBC1, and three heterologous genes: the α-amylase gene from Bacillus licheniformis, the cholesterol oxidase gene from Streptomyces sp., and the lipase gene from Pseudomonas fluorescens. The influence of several fermentative conditions on segregational and structural stability of the recombinant B. stearothermophilus NUB3621 transformants was studied.The parameters of plasmid loss, that is, rate of plasmid loss (R) and specific growth rate difference (δμ), were calculated. B. stearothermophilus NUB3621 carrying pRP9 showed great segregational stability in all the assayed conditions, exceeding more than 300 generations without significant plasmid loss, whereas NUB3621 carrying pPAM2, pPCH2, or pPLY2 exhibited relatively low plasmid stability. The segregational instability of the recombinant constructs increased by increasing the fermentation temperature, decreased by increasing the dilution rate, and was not affected by the level of dissolved oxygen. On the other hand, plasmid maintenance decreased in minimal medium if compared with the results obtained in complex medium. Restriction analyses carried out on cultures of NUB3621 carrying pRP9, pPAM2, pPCH2, or pPLY2, grown for 200 generations on nonselective media, revealed that all the clones tested contained the parental plasmids. These results indicate that the heterologous inserts did not affect the structural stability of the recombinant plasmids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 507-514, 1997.
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  • 16
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    Biotechnology and Bioengineering 54 (1997), S. 153-164 
    ISSN: 0006-3592
    Keywords: hybridoma ; oxygen ; serum-free medium ; continuous culture ; antioxidant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d-1, although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (×18), glutathione S-transferase (×11) and superoxide dismutase (×6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 153-164, 1997.
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  • 17
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 124-128 
    ISSN: 1476-5535
    Keywords: denitrification ; continuous culture ; oxygen ; Comamonas sp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Continuous cultures ofComamonas sp SGLY2 were grown anaerobically prior to establishing steady states at different oxygen flow rates. At a low oxygen transfer rate, no dissolved oxygen accumulated in the medium and all nitrate was reduced to dinitrogen. Concurrently with the increase of dissolved oxygen concentration in the liquid phase, the rate of denitrification decreased. However, at a dissolved oxygen concentration near saturation (33 mg L−1), a part of the electron flow always diverted to nitrate with production of dinitrogen: the aerobic denitrification rate was equivalent to 35% of that calculated under anaerobic conditions. These experiments reflected the co-utilization of oxygen and N-oxides and the production of dinitrogen, up to saturated conditions, which implied synthesis and activity of the four denitrifying enzymes under various aeration conditions.
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  • 18
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    Biotechnology and Bioengineering 49 (1996), S. 185-196 
    ISSN: 0006-3592
    Keywords: streptomycin ; Streptomyces ; strain improvement ; continuous culture ; feedback control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have applied a technique of interactive continuous selection (ICS) to the isolation of streptomycin-resistant mutants of the streptomycin-producing organism, Streptomyces griseus. A series of mutants, each with a different colonial morphology and expressing successively greater resistance to streptomycin, was isolated during the course of selection. Takeover of the mutants has been correlated with changes in on-line estimates of streptomycin concentration such that these estimates may be used as a real-time measure of the genetic state of the cell population. When grown in the medium employed for ICS, mutants expressed increased antibiotic production titers; the best mutant produced 10 to 20 times more streptomycin than the parent strain. Absolute improvements in the maximum specific growth rate and intrinsic resistance to streptomycin did not account for the observed growth advantage of all mutants. Rather, each mutant exhibited relative increases in specific growth rate at increasing concentrations of streptomycin. © 1996 John Wiley & Sons, Inc.
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  • 19
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; hepatitis B surface antigen (HBsAg) ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24s monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24s monomer, is transcribed under the control of the GAL 10p on a chimeric 2-μm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h-1 to washout (0.143 h-1). Both p24s monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24s monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-μm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h-1. Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h-1), even though HBsAg expression was maximal. Total p24s monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h-1. © 1996 John Wiley & Sons, Inc.
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  • 20
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    Biotechnology and Bioengineering 50 (1996), S. 217-221 
    ISSN: 0006-3592
    Keywords: Qβ phage ; molecular evolution ; phage display ; continuous culture ; cellstat ; wall growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lytic coliphage Qβ was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qβ infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. © 1996 John Wiley & Sons, Inc.
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  • 21
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    Biotechnology and Bioengineering 51 (1996), S. 605-612 
    ISSN: 0006-3592
    Keywords: phototrophic bacteria ; Rhodobacter capsulatus ; continuous culture ; light limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of the degree and mode of light limitation on growth characteristics of turbidostat cultures of Rhodobacter capsulatus was investigated using mass and energy balance regularities. Light limitation was achieved by increasing the steady-state biomass concentration at constant incident light intensity (∼100 W/m2) or by decreasing the incident light intensity at constant steady-state biomass concentration (∼500 mg of dry biomass/L). It was shown that under conditions of light limitation of Rh. capsulatus, the content of P and N in the biomass as well as the biomass degree of reduction were determined by the growth rate of the cultures. The energetic yield of biomass of Rh. capsulatus and total bacteriochlorophyll a content increased when light limitation increased. These parameters were higher in the cultures, in which light limitation was achieved by lowering the incident light intensity at low biomass concentration. This seems to be due to different distribution of light within the photobioreactor when dissimilar modes of light limitation were used.
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  • 22
    ISSN: 0006-3592
    Keywords: Methanobacterium thermoautotrophicum ; gaseous substrate limitation ; continuous culture ; mathematical modeling ; amperometric measurement of dissolved H2 concentration ; reaction calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H2, a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from “classic” continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.
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  • 23
    ISSN: 1573-0778
    Keywords: antithrombin III ; mammalian cell culture ; continuous culture ; capillary electrophoresis ; product monitoring ; recombinant protein ; temperature influence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The importance of mammalian cell cultures for biotechnological production processes is steadily increasing, despite the high demands of these organisms on their culture conditions. Efforts towards a more efficient bioprocess generally concentrate on maximizing the culture's life time, the cell number, and the product concentration. Here recombinant BHK 21 c13 cells are used to produce rh-AT III, an anticoagulant of high therapeutic value. The influence of the process mode (batch, repeated batch, continuous perfusion) and the process temperature (30°C vs. 37°C) on the above mentioned parameters is investigated. It is possible to increase the length of the culture from 140 h (batch) to more than 500 h (continuous perfusion culture), while concomitantly increasing the cell density from 0.72 106/ml (batch) to 2.27 106/ml (repeated batch) and 2.87 106/ml (continuous perfusion culture). The accumulation of toxic metabolites, such as lactate, can be curtailed by reducing the bioreactor temperature from 37°C to 30°C during the later part of the exponential growth phase. Fast and reliable product monitoring became essential during process optimization. Capillary zone electrophoresis (CZE) in uncoated fused silica capillaries was studied for that purpose and compared to the standard ELISA. Under optimized conditions an AT III quantification could be done within 2 min with CZE. The detection limit was 5 μg/ml. A relative standard deviation of less than 0.9% was calculated. The detection limit could be lowered by one order of magnitude by using a two dimensional system, where an liquid chromatographic (LC) system is coupled to the CZE. Concomitantly the resolution is improved. The two-dimensional analysis required 5 min. Membrane adsorbers (MA) were used as stationary phase in the LC-system, to allow the application of high flow rates (5–10 ml/min). The correlation between the LC-CZE analysis and the standard AT III-ELISA was excellent, with r2: 0.965. Using the assay for at line product monitoring, it is shown, that the process temperature is of no consequence for the productivity whereas the process mode strongly influences this parameter.
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  • 24
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO cells ; continuous culture ; SV40 promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular β-galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular β-galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.
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  • 25
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    Journal of industrial microbiology and biotechnology 15 (1995), S. 45-48 
    ISSN: 1476-5535
    Keywords: Kluyveromyces fragilis ; lactose transport ; continuous culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V max) are dependent on the dilution rate (D). At D=0.12 h−1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h−1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.
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  • 26
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    World journal of microbiology and biotechnology 11 (1995), S. 643-645 
    ISSN: 1573-0972
    Keywords: Chemostat ; 4-chlorosalicylic acid ; continuous culture ; degradation ; microbial consortium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A mixed, stable microbial community, obtained by continuous enrichment of a sediment core using 4-chlorosalicylic acid as sole source of carbon and energy, contained 10 different bacterial species, including Klebsiella pneumonia, Pseudomonas fluorescens, P. mendocina and P. cichorii. The members of the community were grown separately on various chlorinated compounds which were readily degraded.
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  • 27
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    Biotechnology and Bioengineering 45 (1995), S. 524-535 
    ISSN: 0006-3592
    Keywords: respiration quotient ; carbon dioxide evolution rate ; continuous culture ; cell metabolism ; bicarbonate buffer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The determination of the respiration quotient (RQ = CER/OUR) has not been used so far as a tool for understanding animal cell metabolism. This is due to problems in measuring the carbon dioxide evolution rate (CER) rather than the oxygen uptake rate (OUR). The determination of the CER is complicated by the use of bicarbonate in the medium. Using liquid and gas balances we have derived an equation for continuous culture to quantify the amount of CO2 that comes from the bicarbonate in the feed. Under cell-free conditions, values predicted by this equation agree within 4% with the experimental results. In continuous culture using hybridoma cells, the CO2 from the feed, as determined by an IR-gas analyzer, was found to represent a significant amount of the total measured CO2 in the off-gas (50% in a suboptimal, and 30% in high-growth medium). Furthermore, the problem of CO2 loss from the medium during medium preparation and storage was solved using both a theoretical and an experimental approach. RQ values in continuous culture were evaluated for two different growth media. Small but significant differences in RQ were measured, which were matched by differences in specific antibody rates and other metabolic quotients. In a medium with Primatone RL, an enzymatic hydrolysate of animal cell tissue that causes a more than twofold increase in cell density, the RQ was found to be 1.05, whereas in medium without Primatone RL (but containing amino acids equivalent in composition and concentration to Primatone RL) the RQ was found to be 0.97. We suggest the RQ to be a useful parameter for estimating the physiological state of cells. Its determination could be a suitable tool for both the on-line control of animal cell cultivations and the understanding of cell metabolism. © 1995 John Wiley & Sons, Inc.
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  • 28
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    Biotechnology and Bioengineering 47 (1995), S. 696-702 
    ISSN: 0006-3592
    Keywords: Bacillis subtilis ; spore mutant ; fed-batch ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (〈0.1 h-1) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h-1 the spollG mutant of Bacillus subtilis DB104 (Δnpr Δapr) (Emr) spollG (Bimr):: pMK101 (Cmr) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Δnpr Δapr)::pMK101 (Cmr). © 1995 John Wiley & Sons, Inc.
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  • 29
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    Biotechnology and Bioengineering 47 (1995), S. 520-524 
    ISSN: 0006-3592
    Keywords: bacillus subtilis ; plasmid ; continuous culture ; CAT ; recombinant cultures ; acid formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h-1 production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h-1 were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. © 1995 John Wiley & Sons Inc.
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  • 30
    ISSN: 0006-3592
    Keywords: hybridoma ; subclone ; continuous culture ; batch culture ; igG-mRNA ; biosynthetic activities ; antibody production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: During long-term continuous culture of the hybridoma cell line 11317, a better-producing subclone (I1317-SF11), giving improved productivity, has been selected. The comparison of the original cell line (I1317-DC) with this subclone revealed that although the growth patterns of both clones were similar, both in continuous and in batch cultures, considerable differences could be seen between the clones with respect to monoclonal antibody (MAB) accumulation, MAB production rate, the levels of mRNA coding for heavy and light chains of IgG, and some metabolic activities. In continuous culture as well as in batch culture, I1317-SF11 showed increased levels of mRNA coding for kappa and gamma chains compared with I1317-DC and/or a modified ratio of the mRNA species when compared to that in I1317-DC. Using pulse experiments, it could be established that the biosynthesis of both chains was augmented in I1317-SF11. Although the kappa and gamma mRNA levels were modified or inversed for I1317-SF11, the cells always synthesized more kappa than gamma chains. The overall increase in the synthetic activity of I1317-SF11 is suggested as one reason for the considerable increase of IgG productivity and product accumulation in continuous culture as well as in repeated batch cultures. Tests concerning metabolic activity revealed that I1317-SF11 had a predominantly glycolytic metabolism independent of growth requirements, whereas for I1317-DC the metabolism became increasingly glycolytic with increased growth. The antibody yield coefficient of I1317-SF11 on glutamine was significantly higher than that of I1317-DC for the continuous culture, whereas the antibody coefficients on glucose were almost similar for both clones under the different culture conditions used. Both antibody coefficients were considerablly influenced by the specific growth rate.All these facts together lead to the conclusion that subclone I1317-SF11 uses more of the energy available, or it was the energy and/or precursors available for the synthesis and production of MAB more efficiently than the thesis and production of MAB more efficiently than the original cell line. Although the levels of mRNA coding for heavy and light chains of IgG were modified, it could be confirmed that the overall regulation of MAB-synthesis and -production occurs post-translationally and that at higher growth rates, more biosynthetic activity is diverted to biomass production. © 1994 John Wiley & Sons, Inc.
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  • 31
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    Biotechnology and Bioengineering 43 (1994), S. 471-476 
    ISSN: 0006-3592
    Keywords: hexavalent chromium ; microbial reduction ; Pseudomonas sp. ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Reduction of hexavalent chromium was studied in three bench-scale continuous stirred tank reactors. The inoculum was a culture of Pseudomonas sp., capable of giving 83% to 87% chromate reduction in 72-h batch assays with 60 mg Cr(VI) L-1 in synthetic medium. The continuous culture studies were conducted for about 100 days using synthetic feed containing different levels of chromate (5 to 124 mg L-1) at 28° to 30°C and pH 6.8. The feed rate was varied over the range 0.5 to 1 L d-1 to obtain hydraulic retention time of 36 to 72 h. Chromate reduction efficiency was 81% to 91% and 100% for influent Cr(VI) concentrations of 15 to 124 and 5 mg L-1, respectively, with a hydraulic retention time of 72 h. © 1994 John Wiley & Sons, Inc.
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  • 32
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    Biotechnology and Bioengineering 43 (1994), S. 434-438 
    ISSN: 0006-3592
    Keywords: hybridoma ; continuous culture ; ammonia ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The nature and temporal development of ammonia inhbition were investigated in batch, fed-batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 → 3.7 → 4.0 → 4.9 → 7.7 → 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady-state specific growth kinetics or specific antibody productivity. © 1994 John Wiley & Sons, Inc.
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  • 33
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    Biotechnology and Bioengineering 44 (1994), S. 303-321 
    ISSN: 0006-3592
    Keywords: hybridoma metabolism ; continuous culture ; suspension culture ; antibody productivity ; amino acids ; vitamins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of step-change increase in the concentrations of amino acids and vitamins on the metabolism, growth, and antibody productivity of a murine hybridoma cell line grown in continuous culture on serum-free medium are presented. Additions of the amino acids cysteine with methionine, tryptophan, and isoleucine with valine and vitamin B12 (as cyanocobalamin) resulted in significant increases in viable cell concentrations. Additions of aspartate with asparagine, and threonine with vitamin B1 (as thiamine hydrochloride) resulted in significant increases in final antibody concentrations. Substantial decrease in the fraction of amino acid nitrogen excreted as ammonia occurred upon supplementation with three times the normal concentrations of branched chain amino acids. Decreases in the fraction of amino acid nitrogen converted to ammonia were paralleled by increases in the fraction converted to alanine. © 1994 John Wiley & Sons, Inc.
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  • 34
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    Biotechnology and Bioengineering 41 (1993), S. 43-54 
    ISSN: 0006-3592
    Keywords: acetic acid ; alkaline protease ; Bacilus firmus ; continuous culture ; extracellular enzymes ; carbon/nitrogen/phosphorus limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. © 1993 John Wiley & Sons, Inc.
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  • 35
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    Biotechnology and Bioengineering 42 (1993), S. 1046-1052 
    ISSN: 0006-3592
    Keywords: fungus ; rheology ; morphology ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Aspergillus niger was grown in a 7-L chemostat at biomass levels of 7 to 9 gL-1; dilution rates of 0.03, 0.05, 0.075, and 0.009 h-1; and dissolved oxygen tensions of 7%, 12%, and 40% of air saturation. Broth rheological measurements were made on-line, while off-line image analysis was used to measure mycelial morphology, including characterization of mycelial aggregates (clumps). Under all conditions, more than 87% of the hyphase were in clumps, the shape of which determined the rheological characteristics of the broth. In particular, the power law consistency index could be correlated with the biomass concentration and the roughness factor of the clumps, which describes their hairiness. A decrease in specific growth rate decreased roughness, possibly due to changes in the amount of clump breakup. However, decreases of roughness with increasing dissolved oxygen tension might rather imply some effect on hyphal-hyphal interactions within the clumps. © 1993 John Wiley & Sons, Inc.
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  • 36
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    Biotechnology and Bioengineering 41 (1993), S. 937-946 
    ISSN: 0006-3592
    Keywords: protein excretion ; continuous culture ; Escherichia coli ; β-lactamase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stable continuous overproduction of a plasmidencoded protein, β-lactamase, for at least 50 days by Escherichia coli K-12, RB791(pKN), with release into the culture medium has been demonstrated in two-stage chemostats. The second-stage culture was continuously induced with 0.1 mM IPTG. Continuous expression of β-lactamase could not be sustained with this strain in a single-stage chemostat because of cell death and selection for lac-1 cells. β-Lactamase production in the second stage was sensitive to the second-stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high β-lactamase levels. The best operating conditions found at 20°C were a first-stage dilution rate of 0.12 h-1, a second-stage dilution rate of 0.03 h-1, and equal glucose feed supplied to each stage. Enzymatically active β-lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg β-lactamase/L that was 50% pure at an OD600 〈 6. © 1993 Wiley & Sons, Inc.
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  • 37
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    Biotechnology and Bioengineering 42 (1993), S. 557-570 
    ISSN: 0006-3592
    Keywords: mathematical model of cell growth ; continuous culture ; protein excretion ; β-lactamase ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple mathematical model is developed to help explain the complex population dynamics of an Escherichia coli host-plasmid expression/excretion system for β-lactamase within single- and two-stage reactors. The model successfully integrates the individual regulatory (tac promoter induction), genetic (runaway plasmid replication), and population dynamics (culture instability) aspects of the system. The model predicts, and experiment confirms, that high-level β-lactamase production and excretion cannot be easily maintained in single-stage reactors using the current plasmid construction. Stable target protein production and excretion is mathematically predicted, and experimentally confirmed, within two-stage reactors. The model is used to provide insight into engineering a more stable host-vector expression/excretion system for use in single-stage reactors. © 1993 John Wiley & Sons, Inc.
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  • 38
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    Biotechnology and Bioengineering 41 (1993), S. 572-580 
    ISSN: 0006-3592
    Keywords: phenol degradation ; continuous culture ; Pseudomonas putida ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h-1 and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l-1. These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 974-986 
    ISSN: 0006-3592
    Keywords: hybridoma cell culture ; batch culture ; continuous culture ; integrated product recovery ; monoclonal antibodies ; proteases ; glycosylation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h-1 (experiment 1). However, at a lower dilution rate of 0.015 h-1 (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A280 nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth. © 1993 John Wiley & Sons, Inc.
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  • 40
    ISSN: 1573-0778
    Keywords: continuous culture ; death ; hybridoma ; lactate dehydrogenase ; lysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h−1 to 0.01 h−1 when decreasing the pH from 7 to 6.8.
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  • 41
    ISSN: 1573-0778
    Keywords: antibody production ; continuous culture ; culture age ; hybridoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The stability of the hybridoma cell line MN12 in a long-term homogeneous continuous culture was studied using a panel of analytical methods. These include two flow cytometry methods, for the determination of relative cytoplasmic and membrane IgG content. In addition, the antibody production was determined by an ELISA, and the metabolic state of the cells was determined by means of glucose consumption and lactate production. These results indicate a possible selection of variants of MN12 hybridoma cells with an overall aerobic metabolism, but with a higher glucose consumption rate and a higher lactate production rate. These variants are mainly characterized by a different membrane IgG content and cytoplasmic antibody content. These changes may possibly be affected by the culture age.
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  • 42
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    Biotechnology and Bioengineering 39 (1992), S. 1069-1079 
    ISSN: 0006-3592
    Keywords: carbon dioxide ; bicarbonate ; alkalophilic cultures ; nonideal solutions ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The chemical reactions involving carbon dioxide in mineral culture media are considered. A mathematic model is set up, based on published data, which is valid at pH values below 9, and in which the nonideality of the solution is taken into account. The crucial parameter is the constant expressing the equilibrium between carbon dioxide and bicarbonate, K1.The reactions were studied in three different aqueous solutions: water, mineral salt medium, and a suspension with nongrowing bacterial cells. For each situation, three methods were compared for the determination of the bicarbonate concentration in the solution: equilibrium state total carbon analysis, dynamic monitoring of the rate of acid or alkali addition, and dynamic measurement of the carbon dioxide gas phase mole fraction.In a batch-stirred tank reactor, the equilibrium constant K1 agreed with the published value, and the three bicarbonate analysis methods give the same results. If the nonideality is not taken into account, the result significantly differed from the published value and is likely to be incorrect.A real alkalophilic process, using Acinetobacter calcoaceticus in a continuous stirred tank reactor at steady state, also gave results that are in accord with the literature. However, the results do not allow validation of the equation expressing the nonideality.The steady state in the batch system and in continuous culture can be well described with the mathematical model. However, in the transient state there are some unexplained differences between simulation and measurement.
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  • 43
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    Biotechnology and Bioengineering 40 (1992), S. 1293-1299 
    ISSN: 0006-3592
    Keywords: on-line rheology ; continuous culture ; fungal fermentation ; dissolved oxygen tension ; specific growth rate ; biomass level ; rheology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of oxygen tension, biomass concentration, and specific growth rate on the rheological properties of an Aspergillus niger fermentation broth was investigated by growing the fungus in continuous culture. The rheological properties were measured on-line using an impeller rheometer system. The effect of the specific growth rate on broth rheology was strongly influenced by the dissolved oxygen, (DO) concentration in the broth. At DO concentrations above 10% of saturation, K increased with the dilution rate, and at DO concentrations below 10% of saturation, K decreased with increasing dilution rate. The largest influence of a change in the DO concentration on the viscosity of the broth was found at the lowest growth rates and the lowest DO concentrations. K/x, a term that gives a simple description of the structure or the morphology of the culture, was found to increase with biomass concentration. © 1992 John Wiley & Sons, Inc.
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  • 44
    ISSN: 0006-3592
    Keywords: methylotroph ; continuous culture ; oxiturbidostat ; growth-limiting substrate pulse ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The growth characteristics of a chemostat culture of the obligate methylotrophic bacterium Methylobacillus flagellatum have been determined. Steady-state cultures growing at a rate of 0.73-0.74 h-1, equal to the maximal growth rate, were obtained under oxyturbidostat cultivation conditions. The response of a chemostat culture to a pulse increase of methanol concentration was studied. It was shown that slow and rapidly growing cultures of M. flagellatum responded differently to pulse methanol addition. The growth characteristics of slow-growing cultures decreased after methanol addition compared to those of stationary chemostat cultures. The growth characteristics of rapidly growing cultures were practically unchanged with and without pulse methanol addition.
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  • 45
    ISSN: 0006-3592
    Keywords: Fusarium graminearum ; continuous culture ; chemostat ; morphological mutants ; selection coefficient ; Ks value ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Highly branched (colonial) mutants (MC1-1-, CC1-1, and C106) of Fusarium graminearum A3/5 were each grown with the parental strain (A3/5) in continuous flow cultures at high and low dilution rates using a variety of nutrient limitations. MC1-1 replaced A3/5 in all nutrient-limited cultures tested (glucose-, Mg2+-, ammonium-, and sulphate-limited cultures), suggesting that it has a higher maximum specific growh rate than A3/5. Compared with A3/5, C106 was positively selected for in Mg2+-limited cultures and its selection coefficient was higher at low than at high dilution rates, suggesting that, compared with A3/5, it has a reduced saturation constant (Ks) for Mg2+. However, in batch culture, C106 and A3/5 had the same (15 μM) appaent Ks value for Mg2+. C106 was replaced (negative selection coefficient) by A3/5 in gluose-, ammonium-, and phsophate-limited continuous flow cultures, but was neither at an advantage nor a disadvantage (i.e., it behaved as a neutral mutation) in sulphate-limited cultures. CC1-1 replaced A3/5 when they were grown together in glucose-, maltose-, or ribose-limited continuous flow cultures, but not in fructose-, xylose-, ammonium-, or phsophate-limited cultures. Because A3/5 and CC1-1 had similar Km values (30 μM) for glucose, and because the selective advantage of CC1-1 was maintained in maltose-limited cultures (maltose was not hydrolyzed extracellularly), it was concluded that the selective advantage of CC1-1 did not result from it having a lower Ks for glucose than the parental strain. Rather, the data suggested that the activity of phosphoketopentoepimerase may be altered by the CC1-1 mutation. © 1992 John Wiley & Sons, Inc.
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  • 46
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    Biotechnology and Bioengineering 40 (1992), S. 1243-1255 
    ISSN: 0006-3592
    Keywords: calorimetry, Saccharomyces cerevisiae ; Kluyveromyces fragilis ; yeast metabolism ; modeling ; continuous culture ; respiratory capacity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Yeasts degrade glucose through different metabolic pathways, where the choice of the pathway is dependent on the nature of the limitation in the various substrates. When oxygen is limiting in addition to glucose, yeasts often grow according to a mixture of oxidative and reductive metabolism. Oxygen may be limiting either by supply or by inherent biological restrictions such as the respiratory bottleneck in Saccharomyces cerevisiae or by both. A unified model incorporating both supply and biological limitations is proposed for the quantitative prediction of growth rates, consumption and production rates, as well as key metabolite concentrations during mixed oxidoreductive metabolism occuring as a result of such oxygen limitations. This simple unstructured model can be applied to different yeast strains while at the same time requiring a minimum number of measured parameters. “Estimators” are utilized in order to predict the presence of supply-side or biological limitations. The values of these estimators also characterize the relative importance of oxidative to total metabolism. Results from the aerobic and oxygen-limited chemostat cultures were used to corroborate the model predictions. During these experiments, the heat released by the yeast cultures was also monitored on-line. The model correctly predicted the overall stoichiometry, steady-state concentrations, and rates including heat dissipation rates measured in the various situations of oxygen limitations. Direct continuous measurements such as heat can be used in conjunction with the unified model for on-line proces control. © 1992 John Wiley & Sons, Inc.
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  • 47
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    Biotechnology and Bioengineering 39 (1992), S. 865-869 
    ISSN: 0006-3592
    Keywords: cellulase ; xylose ; Trichoderma reesei ; continuous culture ; growth modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Trichoderma reesei (QM 9414) produced cellulase in continuous culture, on media containing xylose (1%) supplemented with sorbose (0.3%) to induce cellulase production. Maximum cell mass of 4.54 kg/m3 occurred at pH 4.0 and a dilution rate of 0.0391 h-1 where residual substrate was 0.43 kg/m3, but no cellulase was produced. Maximum cellulase production of 0.69 FPU occurred at pH 3.5 and a dilution rate of 0.0110 h-1, where cell mass production was 2.56 kg/m3 and residual substrate was 0.15 kg/m3. Monod kinetic constants, corrected for endogenous metabolism, were 0.091 h-1, 0.469 kg/m3, 0.00923 h-1, and 0.470 kg cells/kg xylose at pH 3.5, for the maximum specific growth rate, Michaelis-Menten coefficient, endogenous metabolism coefficient, and yield coefficient, respectively. Specific growth rate fitted a maturation time model, which predicted decreasing maturation time with increasing pH.
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  • 48
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    Biotechnology and Bioengineering 40 (1992), S. 413-426 
    ISSN: 0006-3592
    Keywords: Bacillus polymyxa ; product profiles ; continuous culture ; bioenergetic model ; oxygen availability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bacillus polymyxa ferments glucose to 1-2,3 butanediol, acetoin, ethanol, acetic acid, lactic acid, and formic acid. This research investigates product formation as a function of oxygen availability. A predictive model that simulates product distribution at known oxygen transfer rates is developed on the hypothesis that, in an energy-limited environment, B. polymyxa utilizes glucose and oxygen in the most efficient manner. The efficiency of utilization of glucose and oxygen is measured in terms of the ATP yields of each oxidative pathway. The identity of the products constituting the profile at the given oxygen transfer rate is determined by comparing the ATP production and consumption rates. While the ATP generated is calculated from a knowledge of the oxygen transfer rate and ATP yields of the oxidative pathways, the ATP consumption is estimated by the Pirt expression in terms of growth- and nongrowth-associated components. The product formation rates are obtained by solving ATP and NAD balance equations. They equate the production and consumption rates of these intermediates and are derived from the pseudo-steady-state hypothesis. The model is applied to continuous culture systems that are both open and closed with respect to biomass. At a given oxygen transfer rate, dilution rate, and inlet glucose concentration, the model predicts steady-state concentrations of two dominant fermentation endproducts with the help of four parameters that can be determined from independent experiments. In contrast with earlier approaches, the experimental studies are carried out in continuous culture. Product profiles are obtained at various oxygen transfer rates, fer rates, inlet glucose concentrations, and dilution rates. The effect of pH on the relative distribution of products is also demonstrated. Results indicate that the model is fairly successful in predicting product profiles as a function of oxygen availability. © 1992 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 39 (1992), S. 504-510 
    ISSN: 0006-3592
    Keywords: hybridoma ; continuous culture ; dialysis ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d-1. Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.
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  • 50
    ISSN: 1573-0778
    Keywords: continuous culture ; free solution capillary electrophoresis ; hollow fibers ; monoclonal antibody characterization ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The performance of a protein-free medium was compared in culture flasks with a serum-supplemented medium and with a serum free medium in terms of cell growth and monoclonal antibody production by a murine hybridoma. We present results of continuous production in hollow fiber culture systems using serum-free medium and protein-free medium. In protein-free medium, it has been possible to produce large quantities of monoclonal antibody with a productivity similar to that obtained in serum-free medium. After a two steps purification process, monoclonal antibodies were characterized by SDS-PAGE, High Performance Size Exclusion Chromatography and Free Solution Capillary Electrophoresis. SDS-PAGE and high performance chromatography analysis have showed that purified monoclonal antibodies produced in serum-free medium or protein-free medium were similar. Furthermore, Capillary Electrophoresis characterization revealed that both MAbs were constituted by three isoforms with equivalent electrophoretic mobilities.
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  • 51
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    Cytotechnology 10 (1992), S. 147-155 
    ISSN: 1573-0778
    Keywords: batch culture ; continuous culture ; hybridoma ; kinetics ; specific rates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.
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  • 52
    ISSN: 0006-3592
    Keywords: catabolite repression ; protein A ; membrane proteins ; continuous culture ; protein expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although widely used in experimental and industrial situations, genetically engineered plasmids containing the lac promoter from Escherichia coli are subject to catabolite repression when grown in glucose-containing media. Several methods of overcoming this problem have been investigated by studying the expression of the protein A gene from Staphylococcus aureus under the control of the Escherichia coli lac promoter. When glycerol is used as a sole carbon source, the plasmid is unstable and is rapidly lost from the culture. When the bacteria are grown in chemostats under glucose limitation, the plasmid is maintained, even at high dilution rates, and the expression of protein A is similar to that observed when glycerol was used. The balance between metabolic load and protein A expression seems to be maintained by reducing the gene dose to a tolerable level. Depending on the metabolic conditions prevailing in the culture, this is achieved, either by reducing the copy number of the plasmid or in extreme cases by removing the plasmid altogether.
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  • 53
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    Biotechnology and Bioengineering 38 (1991), S. 254-259 
    ISSN: 0006-3592
    Keywords: light limitation ; shading ; maintenance ; mathematical model ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Light-limited growth in continuous cultures of phototrophic organisms is modeled. It is assumed that light energy up-take rate depends hyperbolically on light intensity and that the maintenance costs are proportional to biomass. Modeling the light distribution caused by shading within the vessel is necessary to explain the existence of steady state in light-limited chemostats. The model fits well to experimental data from literature on light-limited chemostats and turbidostats. Attention is given to the implications of the model for the estimation of the specific maintenance rate constant in light-limited continuous cultures.
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  • 54
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    Biotechnology and Bioengineering 38 (1991), S. 557-560 
    ISSN: 0006-3592
    Keywords: levan ; continuous culture ; molecular weight ; Erwinia herbicola ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and 13C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.
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  • 55
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    Biotechnology and Bioengineering 38 (1991), S. 972-976 
    ISSN: 0006-3592
    Keywords: cell culture ; antibody production ; fermentation ; continuous culture ; cell growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day-1. The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day-1. Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.
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  • 56
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    Biotechnology and Bioengineering 38 (1991), S. 1020-1028 
    ISSN: 0006-3592
    Keywords: hybridoma ; cell culture ; continuous culture ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A hybridoma cell line, AFP-27-P, was cultivated in continuous culture under glucose-limited conditions. The viable cell concentration, dead-cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP-27-NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod-type function. Furthermore, 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 range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon-growth-associated term and a negative-growth-associated term. The values for the model parameters were determined from regression analysis of the steady state data.
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  • 57
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    Biotechnology and Bioengineering 38 (1991), S. 781-787 
    ISSN: 0006-3592
    Keywords: hybridoma culture ; monoclonal antibody production ; perfusion culture ; continuous culture ; cell cycle ; tangential filtration ; cell separation ; nuclepore membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to elucidate the hybridoma life cycle and the limiting factors in perfusion systems, we performed cultures in a stirred tank bioreactor, coupled to an external tangential flow filtration unit. Cell density and antibody production in perfusion were consistent with previous studies. The average life span of the cells (2.1-2.2 days), antibody, productivity per cell produced (30-38 mg/109 cells) and cell size diameter evolution appeared similar to values observed in batch cultures. These observations highly suggest a similar “grow or die” life cycle. Cell and antibody production, strictly related to the medium perfusion rate, seem to be under the control of the nutrient availability. A hypothesis to explain such a life cycle of hybridoma cells in perfusion systems and a model for viable and dead cell density is proposed.
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  • 58
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    Cytotechnology 5 (1991), S. 201-209 
    ISSN: 1573-0778
    Keywords: continuous culture ; monoclonal antibodies ; passive MCAB release
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed. Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.
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  • 59
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    Cytotechnology 7 (1991), S. 103-112 
    ISSN: 1573-0778
    Keywords: adaptation ; ammonia ; hybridoma ; continuous culture ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH4Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH4Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH4Cl even under the ammonia serial change.
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  • 60
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    Biotechnology and Bioengineering 38 (1991), S. 1271-1279 
    ISSN: 0006-3592
    Keywords: PL promoter ; plasmid content ; 45S RNA ; gene expression ; continuous culture ; two-stage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to investigate how plasmid content, transcription efficiency, and translation efficiency affect the productivity of a cloned gene protein, a new vector (pPLc-RP4.5) was constructed. The vector has PL promoter and lacZ as a structure gene 4.5S RNA gene between PL promoter and lacZ gene. We took advantage of the characteristic that the 4.5S RNA is accumulated inside E. coli cells and can be quantitatively measured. A two-stage continuous culture system in combination with a temperature-sensitive gene switching system was used to study the performance of the recombinant fermentation. It was found that the plasmid content as varied by the dilution rate in the production stage showed a different pattern from that in the growth stage. The result showed that promoter strength had a greater influence on the overall gene expression efficiency of a cloned gene than the plasmid content, and the overall gene expression efficiency was largely dependent upon translation efficiency when a multicopy plasmid (pBR322 derivative and rop-) and a strong promoter (PL) were used to express a heterologous protein in E. coli.
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 75-81 
    ISSN: 0006-3592
    Keywords: mycelial morphology ; Fusarium graminearum ; mycoprtein ; continuous culture ; chemostat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains of Fusarium graminearum, the relatively sparsely branched parental strain (A3/5) and a relatively highly branched “colonial” variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h-1. The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 62
    ISSN: 1573-0778
    Keywords: animal cell culture ; hybridoma ; monoclonal antibody ; packed bed reactor ; continuous culture ; perfusion ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The present work demonstrates the utility of packed bed reactors for the production of monoclonal antibody. We present data from a continuous process run for the production of over 100 grams of antibody, using serum-free medium. An additional pilot run also demonstrates the potential for continued antibody production under protein-free conditions, using a standard basal medium.
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  • 63
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Enzyme and Microbial Technology 15 (1993), S. 652-656 
    ISSN: 0141-0229
    Keywords: Dihydrofolate reductase ; Escherichia coli ; continuous culture ; methotrexate
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 64
    ISSN: 0141-0229
    Keywords: Curve fitting ; antibiotics ; continuous culture ; secondary metabolism
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 65
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Enzyme and Microbial Technology 16 (1994), S. 467-477 
    ISSN: 0141-0229
    Keywords: Catharanthus roseus ; Cinchona robusta ; Hydrodynamic stress ; Nicotiana tabacum ; Tabernaemontana divaricata ; batch culture ; continuous culture ; plant cells
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 66
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Enzyme and Microbial Technology 16 (1994), S. 1079-1083 
    ISSN: 0141-0229
    Keywords: Trichoderma reesei ; cellulase biosynthesis ; comparison with other mutants of T. reesei and soluble substrates ; continuous culture ; growth and maintenance parameters ; lactose
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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