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  • bioreactor
  • continuous culture
  • 1995-1999  (85)
  • 1980-1984  (1)
  • 1925-1929
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 16 (1996), S. 188-191 
    ISSN: 1432-203X
    Keywords: coniferin ; continuous culture ; Linum flavum ; respiration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cell suspension ofLinum flavum was grown in phosphate limited continuous culture at two different growth rates. Energy metabolism (respiration), coniferin and lignin production and overall biomass composition were analysed, in order to establish the relations between growth, maintenance and secondary metabolism. The ATP turnover rate was higher in the faster growing cultures, corresponding with a higher energy requirement. The coniferin production was not directly correlated with the growth rate, indicating the possibility of high production at high growth rates. Steady states grown under identical conditions showed different characteristics that may have evolved during pre-culture time.
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  • 2
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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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  • 3
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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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  • 4
    ISSN: 1573-9368
    Keywords: whey acidic protein gene ; transgenic sheep ; bioreactor ; mammary gland
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.
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  • 5
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    Journal of applied phycology 7 (1995), S. 187-198 
    ISSN: 1573-5176
    Keywords: algal cell culture ; bioreactor ; hydroxy fatty acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min−1 L−1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98μmol photon m−2 s−1) and initial cell density (27 to 149 mg DCW L−1). Maximum cell densities exceeded 1200 mg DCW L−1 after a 20 day cultivation time at optimal conditions of 98μmol photon m−2 s−1 and 118 mg DCW L−1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.
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  • 6
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    Journal of applied phycology 7 (1995), S. 529-537 
    ISSN: 1573-5176
    Keywords: carotenogenesis ; astaxanthin ; light intensity ; continuous culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Under certain culture conditions, cells of the chlorophyte Haematococcus pluvialis accumulate significant amounts of astaxanthin. This study describes biomass and carotenoid production during a sunlight cycle in a continuous culture of growing cells of H. pluvialis and shows that these two parameters are under the control of irradiance. The hourly carotenoid production increases with light intensity and, in our culture conditions, carotenoid accumulation occurs in a few hours and without any morphological change in the algae. These carotenoids seem to be efficient in protecting algal cells against photoinhibition damage if their content is greater than 1% dry biomass. Below this concentration, that is to say in the early hours of high light intensity, dry biomass decreases due to cell lysis. The results demonstrate that secondary carotenoid accumulation in H. pluvialis may occur in the active growth phase and is stimulated from the first hours of sunlight illumination.
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  • 7
    ISSN: 1573-0778
    Keywords: bioreactor ; cell volume ; imaging ; magnetic resonance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Non-invasive magnetic resonance imaging and spectroscopy techniques have been used to monitor the growth and distribution of Chinese hamster ovary K1 cells growing in a fixed bed bioreactor composed of macroporous carriers. Diffusion-weighted 1H magnetic resonance spectroscopy was used to monitor the volume fraction of the bioreactor occupied by the cells and diffusion-weighted 1H magnetic resonance imaging was used to map cell distribution. The imaging measurements demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the surface of the carriers. The increase in the volume fraction occupied by the cells during cell growth showed a close correlation with bioreactor ATP content measured using 31P magnetic resonance spectroscopy. These magnetic resonance measurements, in conjunction with measurements of bioreactor glucose consumption, allowed estimation of the specific glucose consumption rate. This declined during the culture, in parallel with medium glucose concentration.
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  • 8
    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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  • 9
    ISSN: 1573-5176
    Keywords: continuous culture ; cyanobacterium ; Scytonema ; antibiotic ; bioreactor ; immobilization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The filamentous cyanobacterium,Scytonema sp. TISTR 8208, which produces a cyclic peptide antibiotic, was cultivated for 20 d in a seaweed-type bioreactor containing anchored polyurethan foam strips. Cells immobilized onto the foam strips produced the antibiotic for only several days, and the secreted antibiotic disappeared very rapidly from the medium. Cells accumulated the antibiotic intracellularly in a growth-related manner, and secreted it in the stationary phase. Since the antibiotic has a stable physico-chemical nature, the cells seem to take it up and metabolize it. When continuous cultivation was attempted, stable production of the antibiotic was maintained in the bioreactor for 16 d at a dilution rate of 0.01 h−1. Three times more antibiotic was produced in the continuous culture than in the batch culture by the 16th day.
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  • 10
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    Cytotechnology 22 (1996), S. 43-52 
    ISSN: 1573-0778
    Keywords: recombinant CHO cells ; insulin degradative activity ; glycosidase ; bioreactor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two degradative activities were found in a recombinant Chinese hamster ovary cell culture. These activities became more dominant under high cell density and extended running time, as achieved in a semi-continous perfusion culture. The first, insulin degradative activity caused a growth upset in the 3rd cycle of the perfusion culture and shortened the length of the bioreactor process. The second activity, derived from the neutral pH stable sialidase, was found to affect the integrity of the carbohydrate structure of the recombinant protein, causing increase in heterogeneity in molecular weight and pI of the glycoforms. The most efficient way to overcome these problems may be the use of genetically altered ‘designer cells’ as the production cell line.
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  • 11
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    Cytotechnology 22 (1996), S. 111-117 
    ISSN: 1573-0778
    Keywords: aggregation ; bioreactor ; cell growth ; diploid fibroblasts ; microcarriers ; suspension
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Polystyrene microcarriers were prepared in four size ranges (53–63 μm, 90–125 μm, 150–180 μm and 300–355 μm) and examined for ability to support attachment and growth of human diploid fibroblasts. Cells attached rapidly to the microcarriers and there was a direct relationship between cell attachment and microcarrier aggregation. Phasecontrast and scanning electron microscopic studies revealed that while aggregation was extensive, most of the aggregate consisted of void volume. Cell growth studies demonstrated that human diploid fibroblasts proliferated well in microcarrier aggregates, reaching densities of 2.5–3×106 cells per 2 ml dish after 6 days from an inoculum of 0.5×106 cells per dish. When cells were added to the microcarriers at higher density (up to 5×106 cells per 2-ml culture), there was little net growth but the cells remained viable over a 7-day period. In contrast, cells died when plated under the same conditions in monolayer culture. When the microcarriers were used in suspension culture, rapid cell attachment and rapid microcarrier aggregation also occurred. In 100-ml suspension culture, a cell density of 0.7×106 cells per ml was reached after 7 days from an inoculum of 0.1×106 cells. Based on these data, we conclude that microcarrier aggregation is not detrimental to fibroblast growth. These data also indicate that small microcarriers (53–63 μm) (previously thought to be too small to support the growth of diploid fibroblasts) can support fibroblast growth and this occurs primarily because microcarriers in this size range efficiently form aggregates with the cells.
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  • 12
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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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  • 13
    ISSN: 1573-0778
    Keywords: bioreactor ; cord blood ; expansion ; hematopoieticcells ; porous carrier ; stromal cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The ex vivo expansion of hematopoietic progenitor cells is of great interest for a variety of clinical applications, e.g. bone marrow transplantation or gene therapy. Therefore it is of general interest to develop a culture system, able to mimic the in vivo hematopoesis, which is a prerequisite for long-term hematopoietic culture. Our approach was to modify a continuously perfused bioreactor for cultivation and expansion of human hematopoietic stem cells. Therefore we immobilized stromal cells (human primary stromal cells or the murine cell line M2-10B4) in porous glass carriers in a fixed bed reactor and cocultivated human hematopoietic progenitor cells for several weeks. After inoculation of mononuclear cells derived from umbilical cord blood or peripheral blood stem cells both adherent and non adherent cells were harvested and analyzed by flow cytometry and short-term colony assays. During cultivation there was a permanent production of progenitor cells and mature blood cells derived from the immobilized cells in the carriers. We could demonstrate the immobilization of hematopoietic progenitor cells of the myeloid system detectable in short-term colony assays. Additionally we could observe the expansion of very early progenitor cells (CFU-GEMM) up to 4.2-fold and later progenitor cells (CFU-GM and BFU-E) up to 7-fold and 1.8-fold, respectively.
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  • 14
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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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  • 15
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    Cytotechnology 18 (1995), S. 3-8 
    ISSN: 1573-0778
    Keywords: bioreactor ; cellular therapies ; gene therapy ; therapeutic proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Large scale animal cell culture for the production of complex therapeutic proteins has been a major success of the biotechnology industry. Today, approximately half of the $ 5 billion annual turnover of the biotechnology industry is based upon this technology, in many cases with reactors of more than 10 m3. As we look towards the 21 st century, however, we can see novel approaches to the production of therapeutic proteins, by means of gene and cellular therapies. These technologies present new engineering challenges to the animal cell technologist. Are we prepared to meet these challenges? The needs include: small-scale reactors for the preparation of autologous cell lines, methods for the production of viruses to be used as vectors in gene therapy, artificial organ and the processing of xenogenic cell lines and tissues for cellular implants in humans. More attention should be given to three-dimensional cell cultures. Mass transfer considerations need to be extended beyond just oxygen transfer, to include cellular communication in small systems; this is becoming increasingly important for the control and optimise growth and product formation. Apart from improvements of large-scale systems, substantial advantages could be gained by studying new methods for the production and delivery of therapeutic proteins, using small-scale cell culture systems. We should adapt teaching, regulatory, patent and clinical infrastructure to meet this challenge in a harmonious way.
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  • 16
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    Cytotechnology 18 (1995), S. 27-34 
    ISSN: 1573-0778
    Keywords: Air lift reactor ; bubble column ; bioreactor ; oxygen gradients ; scale-up ; stirred vessel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An estimation is made of oxygen gradients in animal-cell bioreactors, using straightforward engineering calculations. Three types of bioreactor are considered: stirred vessel, bubble column and air lift, of sizes between 0.01 and 10 m3. First, the gradient is estimated in the stagnant layer surrounding a cell (15 μm), a microcarrier (185 μm) with 300 cells attached to it, a macroporous support (1.25 mm) containing 185,00 cells and one (6 mm) containing 4.25 million cells. It is assumed that oxygen consumption is 10−16 mole O2·cell−1·s−1, while mass transfer coefficients are obtained from Sherwood relations. Circulation and liquid-retention times of the bioreactors are compared with the oxygen-exhaust times of suspensions with 1012, 1013 and 1014 cells/m3 to estimate if oxygen gradients are likely to exist in the bulk-liquid phase. Finally, the gradient in the liquid film surrounding air bubbles is estimated using k l A-values obtained from empirical correlations. It is clear from all these estimations that in many situations severe gradients can be expected. The question remains, however, whether gradients should be avoided as much as possible, or may be tolerated to a certain extent or even created on purpose because of possible beneficial effects.
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  • 17
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    Cytotechnology 18 (1995), S. 57-66 
    ISSN: 1573-0778
    Keywords: Automation ; bioreactor ; optimisation ; process control ; software sensors ; validation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The Batch Control System for Vaccines (BCSV), a new Man Machine Interface (MMI) for the control of cultivations in bioreactors, was developed according to SP-88. SP-88 is the ISA standard for Batch Control Systems. Among others, SP-88 supplied the concept of recipes, which organize and specify the monitoring and control requirements for manufacturing. Process optimisation and compliance to GMP rules and regulations were the main objectives for this development. The most important features of the BCSV interface include: - implementation at production, pilot and R & D scale to assure easy transfer of knowledge and experience at the various stage of process development; - independency of underlying hardware to ensure similar “look and feel” for different pieces of equipment; - in-house development and maintenance of recipes to have maximum control over applications; - interactive communication between operator and BCSV during recipe execution. GMP compliance was assured not only by considering governing sets of GMP regulations, but also by taking up the interface in a overall Information & Automation strategy and by setting up a QA strategy for the entire life cycle of the system.
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  • 18
    ISSN: 1573-0778
    Keywords: ex vivo expansion ; hematopoietic culture ; bioreactor ; clinical therapies ; cytokines ; stroma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34+ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.
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  • 19
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    Cytotechnology 30 (1999), S. 169-172 
    ISSN: 1573-0778
    Keywords: adenovirus ; bioreactor ; microcarriers ; serum-free medium ; 293 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor.
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  • 20
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    Neural processing letters 4 (1996), S. 107-112 
    ISSN: 1573-773X
    Keywords: bioreactor ; generalized predictive control ; neural networks ; nonlinear systems
    Source: Springer Online Journal Archives 1860-2000
    Topics: Computer Science
    Notes: Abstract This paper presents the Generalized Predictive Control (GPC) strategy based on Artificial Neural Network (ANN) plant model. To obtain the step and the free process responses which are needed in the generalized predictive control strategy we iteratively use a multilayer feedforward ANN as a one-step-ahead predictor. A bioprocess was chosen as a realistic nonlinear SISO system to demonstrate the feasibility and the performance of this control scheme. A comparison was made between our approach and the adaptive GPC (AGPC).
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  • 21
    ISSN: 1573-5117
    Keywords: continuous culture ; high density culture ; marine rotifers ; Brachionus rotundiformis ; Brachionus plicatilis ; mass production ; nutritionalimprovement
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to improve culture stability, as well as to reduce labor andspace for mass production of the marine rotifer Brachionus, an automaticcontinuous culture system has been developed for practical use. The systemconsists of a filtration unit, a culture unit and a harvest unit. In thissystem, filtered water and food are continuously supplied into a rotiferculture tank at a pre-determined rate, and the same amount of culture wateris transferred into a harvest tank to obtain rotifers at a significantbiomass. B. rotundiformis (so-called S-type) and B. plicatilis (so-calledL-type) were successfully mass-produced from the system, under regulatedconditions of 30 °C (for S-type) or 24°C (for L-type), 20 ppt diluted sea water and totaldarkness. As food for rotifers, commercially available concentrated freshwater Chlorella vulgaris was used. The dilution rate was adjusted to0.6–0.7 d−1 for S-type rotifer and 0.25d−1 for L-type rotifer, respectively. The average productionwas about 2.1 billion rotifers d−1 from a 1-m3S-type continuous culture in which high rotifer densities ranging from 3000to 6000 ind. ml−1 were maintained. On the other hand, about0.17 billion rotifers d−1 on average were obtained from a500-liter L-type culture, with densities ranging from 1100 to 2200 ind.ml−1. The longest duration for a continuous culture was over110 days. Furthermore, experiments utilizing five enrichment techniquesindicate that rotifers from the continuous culture system can easily benutritionally enriched in the same manner as those from common batch culturesystems.
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  • 22
    ISSN: 1573-5117
    Keywords: deep seawater ; benthic diatom ; Nitzschia sp. ; continuous culture ; bioreactor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A continuous culture system for a benthic food diatom Nitzschia sp. wasestablished by using properties of high nutrient and clean of deep seawater(DSW). DSW collected from 320 m depth in Muroto City, Japan, was introducedinto a glass-pipe bioreactor (14 cm length, 3 cm diam.) containing glassbeads of 0.5 cm diam. as substrata for the alga, and it was incubated at18°C · 80μEm−2sec−1 · L:D=14:10. The chlorophyll a yield of benthicdiatoms in a reactor as a unit of surface area of the substratum was only0.001–0.003 μg cm−2 when the flow rate of DSW was 0(batch culture conditions). However, when DSW was supplied continuously to areactor, the yield increased to 1.4 μg-chl.a cm−2 alongwith the increase in flow rate of DSW. Moreover, amounts of chl.a washed outof the system were negligible, 0.0014 to 0.0045%, even though theflow rate of DSW was as much as 25 times h−1, suggesting thatsloughing of benthic diatoms from the substratum was minimized. Although theyield of diatoms fluctuated significantly at the time that the DSW wascollected, the variation could be minimized by increasing the flow rate ofDSW. These results indicate that the continuous culturing system with DSWsupports the stable and effective mass culture of benthic food diatom.
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  • 23
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    Evolutionary ecology 12 (1998), S. 153-164 
    ISSN: 1573-8477
    Keywords: algae ; anti-predator adaptations ; arms race ; Chlorella vulgaris ; chrysophytes ; continuous culture ; evolution ; flagellates ; multicellularity ; Ochromonas vallescia ; phagocyte ; predator–prey interactions ; selective pressure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Predation was a powerful selective force promoting increased morphological complexity in a unicellular prey held in constant environmental conditions. The green alga, Chlorella vulgaris, is a well-studied eukaryote, which has retained its normal unicellular form in cultures in our laboratories for thousands of generations. For the experiments reported here, steady-state unicellular C. vulgaris continuous cultures were inoculated with the predator Ochromonas vallescia, a phagotrophic flagellated protist (‘flagellate’). Within less than 100 generations of the prey, a multicellular Chlorella growth form became dominant in the culture (subsequently repeated in other cultures). The prey Chlorella first formed globose clusters of tens to hundreds of cells. After about 10–20 generations in the presence of the phagotroph, eight-celled colonies predominated. These colonies retained the eight-celled form indefinitely in continuous culture and when plated onto agar. These self-replicating, stable colonies were virtually immune to predation by the flagellate, but small enough that each Chlorella cell was exposed directly to the nutrient medium.
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    Plant cell, tissue and organ culture 42 (1995), S. 81-90 
    ISSN: 1573-5044
    Keywords: Adhesion ; bioreactor ; immobilization ; plant cell culture ; secondary metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.
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    Plant cell, tissue and organ culture 43 (1995), S. 123-126 
    ISSN: 1573-5044
    Keywords: bioreactor ; dissolved oxygen ; haploid cell suspension ; pH ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Maize (Zea mays L.) haploid cells were cultivated in a 1500 ml aerated and stirred batch bioreactor using modified BM medium. Cell growth was highly affected by pH and dissolved oxygen, and we observed two fairly distinct growth phases. During the first two days after inoculation at pH 5.8, oxygen consumption was high and the cells lowered the pH to a value around 4.3. After this period the pH stabilized at 4.5 and the dissolved oxygen reached a steady level. Decreasing dissolved oxygen concentration leads to lower growth rate and to higher pH. Both events mean stress conditions for the cell culture and probably result in increased genetic variability, and the loss of regeneration capacity. The stress condition during the adaptation phase can be eliminated by decreasing the pH of the medium to 4.7 before inoculation and by keeping dissolved oxygen above 40%. These conditions provide prolonged exponential growth dynamics and the cell suspensions could be the basis of large scale cultures also.
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    Plant cell, tissue and organ culture 57 (1999), S. 29-38 
    ISSN: 1573-5044
    Keywords: bioreactor ; carrot ; dissolved oxygen ; plant micropropagation ; somatic embryo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To evaluate the relationship between somatic embryogenesis and dissolved oxygen concentration, somatic embryo cultures of carrot (Daucus carota L.) were cultured under various dissolved oxygen concentration levels (bubble free aeration with 4%, 7%, 20%, 30%, and 40% oxygen in flasks). The system used allows dissolved oxygen concentration control without bubble aeration or mixing speed modification. The total number of somatic embryos was not affected by the dissolved oxygen (DO) concentration tested. Even if globular-stage embryos were induced at a low level of oxygen aeration, heart-stage embryo formation was still repressed. Oxygen enrichment (20%, 30% and 40% oxygen) enhanced torpedo and cotyledonary-stage embryo production. The oxygen-enriched aeration was effective in promoting the growth of the late developmental stages. Sugar consumption did not increase when the oxygen concentration was enriched above the ambient level. The number of heart-stage embryos increased as oxygen concentration increased up to the 7% level, while above the 20% level no change in production was observed. The production of cotyledonary-stage embryos was directly related to oxygen concentration. These results support that oxygen-enriched aeration provides oxygen to the low oxygen areas in somatic embryo. After the heat-stage embryos, which were grown at the 7% level were transferred to a flask with ambient, they developed an elongated root part and eventually grew to normal plantlets.
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  • 27
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 22-28 
    ISSN: 1476-5535
    Keywords: lipase ; recombinantXanthomonas ; fed-batch ; bioreactor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Recombinant plasmid pBP13, which expresses the alkaline lipase fromPseudomonas aeruginosa IGB83 under thetac promoter was transferred toXanthomonas campestris pvcampestris IBT148. Different fermentation conditions were tested for lipase productivity by strain IBT148 carrying plasmid pBP13, and a fermentation process was established in an instrumented bioreactor, where lipase production was increased more than 12-fold with respect to the initial culture conditions in shake flasks. Xanthan gum stabilized the activity of the alkaline lipase.
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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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  • 29
    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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    World journal of microbiology and biotechnology 14 (1998), S. 685-688 
    ISSN: 1573-0972
    Keywords: Antibiotic ; bioreactor ; Chromobacterium violaceum ; pigment ; violacein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A procedure for the production, extraction, and purification of violacein was developed using Chromobacterium violaceum (CCT 3496) cultivated on cotton, in modified 1 litre Roux bottles. A surface tray bioreactor was built to perform these experiments. Violacein was extracted with commercial ethanol, and purified by filtration, Soxhlet extraction, crystallization and high performance liquid chromatography. The violacein was analysed and identified by proton and carbon-13 NMR spectroscopies, thermogravimetric analysis, mass spectrometry, UV-VIS spectroscopy and infrared spectroscopy. It was concluded that the product was highly purified violacein.
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    Antonie van Leeuwenhoek 74 (1998), S. 107-118 
    ISSN: 1572-9699
    Keywords: amidase ; biocatalyst ; bioreactor ; nitrilase ; polyacrylamide ; Rhodococcus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present. The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems. Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.
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    Biodegradation 6 (1995), S. 39-46 
    ISSN: 1572-9729
    Keywords: oxygen tension ; 2,5-dichlorobenzoic acid ; mixed substrates ; continuous culture ; adaptation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract From long-term chemostat experiments, variants ofPseudomonas aeruginosa JB2 were obtained which exhibited altered properties with respect to the metabolism of 2,5-dichlorobenzoic acid (2,5-DBA). Thus, unlike the original strain JB2-WT, strain JB2-var1 is able to grow in continuous culture on 2,5-DBA as the sole limiting carbon and energy source. Yet, at a dilution rate of 0.07 h−1 and a dissolved oxygen concentration of ≤ 12 µM, even with this strain no steady states with 2,5-DBA alone could be established in continuous cultures. Yet another strain was obtained after prolonged continuous growth of JB2-var1 in the chemostat. It has improved 2,5-DBA degrading capabilities which become apparent only during growth in continuous culture: a lower apparent K m for 2,5-DBA and lowered steady-state residual concentrations of 2,5 DBA. Although with this strain steady states were obtained at oxygen concentrations as low as 11 µM, at further lowered concentrations this was no longer possible. In C-limited continuous cultures of JB2-var1 or JB2-var2, addition of benzoic acid (BA) to the feed reduced the amounts of 2,5-DBA degraded, which was most apparent at low oxygen concentrations (〈 30 µM). At higher dissolved oxygen concentrations the addition of BA resulted in increasing cell-densities but did not affect the residual steady state concentration of 2,5-DBA. Indeed, whole cell suspensions from chemostat cultures grown on BA plus 2,5-DBA did show a lower apparent affinity for 2,5-DBA than those from cultures grown on 2,5-DBA alone. These results indicate that in environments with low oxygen concentrations and alternative, more easily degradable, substrates the degradation rates of chloroaromatic compounds by aerobic organisms may be negatively affected.
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    Biodegradation 10 (1999), S. 219-233 
    ISSN: 1572-9729
    Keywords: bioavailability ; biodegradation ; bioreactor ; biotreatment ; NAPL ; toxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Two-liquid-phase culture systems involve the addition of a water-immiscible, biocompatible and non-biodegradable solvent to enhance a biocatalytic process. Two-liquid-phase bioreactors have been used since the mid-seventies for the microbial and enzymatic bioconversion of hydrophobic/toxic substrates into products of commercial interest. The increasing popularity of bioremediation technologies suggests a new area of application for this type of bioreactor. The toxicity and the limited bioavailability of many pollutants are important obstacles that must first be overcome in order to improve biodegradation processes. Two-liquid-phase bioreactors have the potential to resolve both limitations of biotreatment technologies by the enhancement of the mass-transfer rate of compounds with low bioavailability, and by the controlled delivery of apolar toxic compounds. This technology can also be useful in accelerating the enrichment of microorganisms degrading problematic pollutants. In this paper, we discuss the application of two-liquid-phase bioreactors to enhance the biodegradation of toxic/poorly bioavailable contaminants. Important microbial mechanisms involved in this type of system are described. Uptake of the substrates can be achieved by microorganisms freely dispersed in the aqueous phase and/or bound at the interface between the aqueous and the immiscible phases. Production of surface-active compounds and adhesion abilities are microbial features involved in the process. General guidelines for the design of two-liquid-phase bioreactors for biodegradation purposes are presented. Solvent selection should be established on specific criteria, which depend on the characteristics of target compound(s) and the microorganism(s) implicated in the biodegradation process. The central importance of maximizing the interfacial surface area is highlighted. The potential of this approach as an alternative to current biotreatment technologies is also discussed.
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  • 34
    ISSN: 1573-5117
    Keywords: Rotifera ; Brachionus ruhens ; Brachionus calyciflorus ; population dynamics ; steady-state growth ; continuous culture ; size distributions
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    Topics: Biology
    Notes: Abstract In laboratory studies, rotifers (Brachionus calyciflorus) were monitored under well-defined environmental conditions at different supply rates of a unicellular algal food (Chlorella vulgaris). Rotifer size frequency distributions are described for conditions of steady-state growth, exponential increase, and starvation. Temporal fluctuations in size-age structure are described for cultures during transient conditions during the approach to a steady state and following step changes in food supply rate. The size structures of the populations displayed definite and reproducible shifts among typical patterns during transient conditions, reflecting the physiological and other dynamic processes that underlay the population dynamics. Size structure probably is a key variable that should be included in models for predicting growth dynamics during transient growth conditions.
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  • 35
    ISSN: 1573-5044
    Keywords: atmosphere ; bioreactor ; Ipomoca batatas ; somatic embryogenesis ; suspension culture
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    Topics: Biology
    Notes: Abstract Somatic embryos of Ipomoea batatas Lam. (sweet potato cv. ‘White Star’) were produced in an airlift bioreactor. This work describes the optimization of the embryogenic system on semisolid medium, followed by transfer of the system to liquid cultures and ultimately to the airlift bioreactor. The physiological age of embryogenic callus influenced the number and overall morphology of the embryo population in both semisolid and liquid medium. Maximum mature embryo production (35 embryos 10 mg-1 inoculum) was obtained from six-week-old callus at 30°C. Somatic embryogenesis occurred in liquid cultures containing 20 mM NH4NO3 and 30 mM KCl. Globular embryos formed and continued development in suspension producing viable, mature cotyledonary embryos by day 14. Embryo formation and development was limited in the bioreactor. Although shear stress was responsible for some embryogenic damage, the effect of purging the system with fresh air needed to be investigated. To isolate aeration effects from shear stress effects, atmospheric determinations were performed on shaker flask cultures. Initially the gas composition within the Erlenmeyer headspace was that of room air. Ethylene increased to a maximum of 6.4 ppm (day 16), maximum CO2, 21.2%, was also evident on day 16, and oxygen was depleted to a minimum of 8.1% by day 14. Purging the cultures with fresh air reduced the number of embryos formed; however, they were significantly longer than those formed in closed flasks. The gas response model of Ipomoea batatas will enable atmosphere replenishment in the bioreactor mimicking that of the shaker flask environment. Once the damaging effects of shear stress have been overcome, the regulation of bioreactor gasses should allow somatic embryo formation in the bioreactor comparable to that in shaker flasks.
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    Plant cell, tissue and organ culture 45 (1996), S. 277-280 
    ISSN: 1573-5044
    Keywords: bioreactor ; contamination ; medium filtration ; micropropagation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Contaminated Syngonium clusters were multiplied in an air lift bioreactor in liquid medium containing sucrose with the medium being circulated through a sterilizing filter. After 30 days, the culture in filtered medium produced 19.5 shoot initials per gram fresh weight of inoculum compared to 8.7 shoot initials produced in unfiltered medium. Transfer to an elongation medium with 30 mg l-1 Rifampicin produced shoots on 67% of the clusters, while transfer to elongation medium without Rifampicin poduced shoots on 40% of the clusters. Clusters grown for three subcultures in a reactor without medium filtration had lost their multiplication ability. Clusters grown for three subcultures in a reactor with filtration, however, continued to show a two-three fold increase in fresh weight and shoot production.
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  • 37
    ISSN: 1573-5087
    Keywords: medium exchange ; plant cell suspension culture ; bioreactor ; somatic embryogenesis ; Picea sitchensis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer. The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions. The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.
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  • 38
    ISSN: 1573-5117
    Keywords: Rotifera ; Brachionus ruhens ; Brachionus calyciflorus ; population dynamics ; steady-state growth ; continuous culture ; size distributions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In laboratory studies, rotifers (Brachionus calyciflorus) were monitored under well-defined environmental conditions at different supply rates of a unicellular algal food (Chlorella vulgaris). Rotifer size frequency distributions are described for conditions of steady-state growth, exponential increase, and starvation. Temporal fluctuations in size-age structure are described for cultures during transient conditions during the approach to a steady state and following step changes in food supply rate. The size structures of the populations displayed definite and reproducible shifts among typical patterns during transient conditions, reflecting the physiological and other dynamic processes that underlay the population dynamics. Size structure probably is a key variable that should be included in models for predicting growth dynamics during transient growth conditions.
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  • 39
    ISSN: 1573-5117
    Keywords: continuous culture ; freshwater sponge ; Ephydatia fluviatilis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The freshwater sponge, Ephydatia fluviatilis (Porifera: Spongillidae), was maintained in a continuous-flow laboratory culture system under several conditions of calcium ion (Ca++) concentration and salinity. Experimental results suggest that sponge growth rate increases with increasing Ca++ concentration, that sponge growth rate decreases with increasing salinity, and that the negative effect of higher salinity can be overcome by increasing Ca++ concentration. The experimental results correlate well with field observations on the effects of salinity and Ca++ on the distribution of E. fluviatilis.
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  • 40
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    Biotechnology and Bioengineering 59 (1998), S. 351-359 
    ISSN: 0006-3592
    Keywords: bioreactor ; high density ; insect cells ; perfusion ; Sf9 ; ultrasonic filter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high-density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high-density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte-colony inhibition factor. The protein yield on a cell basis from infecting high-density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high-density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high-density perfusion culture for large-scale protein expression from Sf9 cells are also highlighted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:351-359, 1998.
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    Biotechnology and Bioengineering 50 (1996), S. 36-48 
    ISSN: 0006-3592
    Keywords: insect cell culture ; Sf-9 cells ; respiration ; bioreactor ; on-line monitoring ; baculovirus expression vector system ; recombinant proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Respiration rates in Spodoptera frugiperda (Sf-9) cell bioreactor cultures were successfully measured on-line using two methods: The O2 uptake rate (OUR) was determined using gas phase pO2 values imposed by a dissolved oxygen controller and the CO2 evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf-9 cultures. Infection led to increases in volumetric and per-cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant β-galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5-100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant β-galactosidase. © 1996 John Wiley & Sons, Inc.
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  • 42
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    Biotechnology and Bioengineering 56 (1997), S. 1-8 
    ISSN: 0006-3592
    Keywords: transesterification ; hydrolysis ; water activity ; cutinase ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.
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  • 43
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    Cytotechnology 20 (1996), S. 191-198 
    ISSN: 1573-0778
    Keywords: insect cell culture ; perfusion culture ; membrane perfusion ; crossflow microfiltration ; baculovirus ; bioreactor ; fluidized bed ; packed bed ; recombinant protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates. For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.
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    Cytotechnology 26 (1998), S. 227-236 
    ISSN: 1573-0778
    Keywords: bioreactor ; computer control ; data acquisition ; glucose control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A general approach is described for the implementation of a networked multi-unit computer integrated control system. The use of data acquisition hardware and graphical programming tools alleviates tedious programming and maintains potency and flexibility. One application of the control system, the control of a mammalian cell perfusion culture based on a key nutrient glucose concentration, was demonstrated. The control system offers customized user interface for all process control parameters and allows the flexibility for continued improvement and implementation of new tailored functions. The temperature, pH, dissolved oxygen and glucose level were accurately controlled.
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  • 45
    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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  • 46
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    Cytotechnology 29 (1999), S. 135-149 
    ISSN: 1573-0778
    Keywords: anchorage dependent ; animal cell ; bioreactor ; Cultispher S ; Cytodex 3 ; microcarrier ; respiratory syncytial virus ; vaccine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Veterinary viral vaccines generally comprise either attenuated or chemically inactivated viruses which have been propagated on mammalian cell substrates or specific pathogen free (SPF) eggs. New generation vaccines include chemically inactivated virally-infected whole cell vaccines. The NM57 cell line is a bovine nasal turbinate persistently infected (non-lytic infection) with a strain of the respiratory syncytial virus (RSV). The potential of microcarrier technology for the cultivation in bioreactors of this anchorage dependent cell line for RSV vaccine production has been investigated. Both Cytodex 3 and Cultispher S microcarriers proved most suitable from a selection of microcarriers as growth substrates for this NM57 cell line. Maximum cell densities of 4.12×105 cells ml-1and 5.52×105 cells ml-1 respectively were obtained using Cytodex 3 (3 g l-1) and and Cultispher S (1 g l-1) in 5 l bioreactor cultures. The fact that cell growth was less sensitive to agitation rate when cultured on Cultispher S microcarriers, and that cells were efficiently harvested from this microcarrier by an enzymatic method, suggested Cultispher S is suitable for further evaluation at larger bioreactor scales (〉5 l) than that described here.
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  • 47
    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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  • 48
    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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  • 49
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    Cytotechnology 30 (1999), S. 149-158 
    ISSN: 1573-0778
    Keywords: bioreactor ; cell culture ; disposable ; wave agitation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications.
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  • 50
    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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  • 51
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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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  • 52
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    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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  • 53
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    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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  • 54
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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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  • 55
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    Biotechnology and Bioengineering 45 (1995), S. 387-397 
    ISSN: 0006-3592
    Keywords: transesterification ; water activity ; lipolytic enzymes ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (aw). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters. © 1995 John Wiley & Sons, Inc.
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  • 56
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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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  • 57
    ISSN: 0006-3592
    Keywords: hollow fiber ; bioreactor ; immobilized enzymes ; porosity ; phospholipase A2 ; low densitylipoprotein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Previous studies have shown that the modification of low density lipoprotein (LDL) by the enzyme phospholipase A2(PLA2)results in a reduction of cholesterol levels in the plasma of hypercholesterolemic rabbits, due to accelerated clearance of the modified LDL. In the current study, we established techniques and optimized the ratio of enzyme to support for the immobilization of PLA2 on a polymeric support. Hollow fiber bioreactors made from polytetrafluoroethylene (PTFE) polymers were used to encapsulate immobilized PLA2. This design was adopted to eliminate hemolysis of red blood cells by the enzyme. Characterization of the resulting immobilized enzyme in terms of its activity, Michaelis-Menten kinetic constants, and the variation of its activity with incubation time is presented. The enzyme activity was not significantly altered upon incubation at 37°C in lipoprotein-deficient serum (LPDS), over the course of 2 months. The Michaelis-Menten kinetics constants are KM = 8.9 mM, Vmax = 6434.2 for the free enzyme and KappM = 16.7 mM, Vappmax = 619.7 for the immobilized enzyme. These data suggest that a system based on immobilized PLA2 in conjunction with hollow fiber bioreactors (HFBs) may be a good candidate for lowering LDL levels in plasma. © 1995 John Wiley & Sons, Inc.
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  • 58
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    Biotechnology and Bioengineering 47 (1995), S. 384-400 
    ISSN: 0006-3592
    Keywords: hollow fiber ; bioreactor ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.© John Wiley & Sons, Inc.
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  • 59
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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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  • 60
    ISSN: 1573-9368
    Keywords: bioreactor ; gene farming ; genetic engineering ; mammary gland ; milk composition ; recombinant protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 ± 12μg/ml (ranging from 223 ± 61 to 484 ± 39 μg/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 ± 41 μg/ml (ranging from 360 ± 15 to 678 ± 80 μg/ml). Homozygous huIGF-1 females clearly showed a significantly increased production performance of the recombinant protein.
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  • 61
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    Biotechnology and Bioengineering 49 (1996), S. 78-86 
    ISSN: 0006-3592
    Keywords: crossflow microfiltration ; hydrodynamics ; fouling ; bioreactor ; Lactobacillus helveticus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A filtration rig equipped with a tubular alumina membrane was used to study the performance of crossflow microfiltration of Lactobacillus helveticus. Experiments were performed at constant permeation flux. High cell concentrations and fast transient conditions to the stationary J adversely affected permeability. Membrane fouling was due to a fast irreversible layer formation and to a reversible cell cake. This microbial deposit characteristics were dependent on the ratio permeation flux/wall shear stress, J/τw. Fouling was faster and more severe when J/τw was greater than a critical value of 1.15 L-1 · h-1 · m-2 · Pa-1. The disordered structure of this cell cake seemed to lead to a macromolecule deposit between the cells which adversely affected the membrane permeability. © 1996 John Wiley & Sons, Inc.
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  • 62
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    Biotechnology and Bioengineering 50 (1996), S. 479-492 
    ISSN: 0006-3592
    Keywords: stem cell ; bioreactor ; stromal layer ; Graetz number ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10-1, 100, and 101) and P(10-2, 10-1, 100, 101, and 102). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, …, 0.1 and Gz = 0.1, …, 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions. © 1996 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 52 (1996), S. 34-44 
    ISSN: 0006-3592
    Keywords: hepatocyte spheroid ; porcine hepatocyte ; hollow fiber ; bioartificial liver ; collagen ; bioreactor ; ureagenesis ; albumin synthesis ; glucuronidation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A xenogeneic hollow fiber bioreactor utilizing collagen-entrapped dispersed hepatocytes has been developed as an extracorporeal bioartificial liver (BAL) for potential treatment of acute human fulminant hepatitis. Prolonged viability, enhanced liver-specific functions, and differentiated state have been observed in primary porcine hepatocytes cultivated as spheroids compared to dispersed hepatocytes plated on a monolayer. Entrapment of spheroids into the BAL can potentially improve performance over the existing device. Therefore, studies were conducted to evaluate the feasibility of utilizing spheroids as the functionally active component of our hybrid device. Confocal microscopy indicated high viability of spheroids entrapped into cylindrical collagen gel. Entrapment of spheroids alone into collagen gel showed reduced ability to contract collagen gel. By mixing spheroids with dispersed cells, the extent of collagen gel contraction was increased. Hepatocyte spheroids collagen-entrapped into BAL devices were maintained for over 9 days. Assessment of albumin synthesis and ureagenesis within a spheroid-entrapment BAL indicated higher or at least as high activity on a per-cell basis compared to a dispersed hepatocyte-entrapment BAL device. Clearance of 4-methylumbelliferone to its glucuronide was detected throughout the culture period as a marker of phase II conjugation activity. A spheroid-entrapment bioartificial liver warrants further studies for potential human therapy. © 1996 John Wiley & Sons, Inc.
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  • 64
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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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  • 65
    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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  • 66
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    Biotechnology and Bioengineering 55 (1997), S. 163-169 
    ISSN: 0006-3592
    Keywords: bioreactor ; paint stripper solvents ; biodegradation ; model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Paint stripping wastes generated during the decontamination and decommissioning of former nuclear facilities contain paint stripping organics (dichloromethane, 2-propanol, and methanol) and bulk materials containing paint pigments. It is desirable to degrade the organic residues as part of an integrated chemical-biological treatment system. We have developed a modified gas lift loop bioreactor employing a defined consortium of Rhodococcus rhodochrous strain OFS and Hyphomicrobium sp. DM-2 that degrades paint stripper organics. Mass transfer coefficients and kinetic constants for biodegradation in the system were determined. It was found that transfer of organic substrates from surrogate waste into the air and further into the liquid medium in the bioreactor were rapid processes, occurring within minutes. Monod kinetics was employed to model the biodegradation of paint stripping organics. Analysis of the bioreactor process was accomplished with BIOLAB, a mathematical code that simulates coupled mass transfer and biodegradation processes. This code was used to fit experimental data to Monod kinetics and to determine kinetic parameters. The BIOLAB code was also employed to compare activities in the bioreactor of individual microbial cultures to the activities of combined cultures in the bioreactor. This code is of benefit for further optimization and scale-up of the bioreactor for treatment of paint stripping and other volatile organic wastes in bulk materials. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 163-169, 1997.
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  • 67
    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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  • 68
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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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  • 69
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    Biotechnology and Bioengineering 51 (1996), S. 131-140 
    ISSN: 0006-3592
    Keywords: mixing power ; convection ; fermentation ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The phenomena of mixing and mass transfer of substrates to microorganisms greatly affect the biochemical reactions which take place in fermentation processes. The effect that agitation power has on the observable reaction kinetics involved in beer fermentation has been studied in different types of bioreactors, from laboratory to industrial scale. With this aim in mind, an effectiveness factor, η, is introduced which is defined as the relation between the existing rate of reaction, whichever bioreactor is considered, and the reaction rate in the well-mixed, and therefore presumably homogeneous, bioreactor with no diffusional limits. The limitation to homogeneously supplying nutrient material to the cells produces a decrease in this effectiveness factor, which has been correlated to the energy dissipation rate with a similar slope to that which appears in an existing correlation in the literature between this energy and the mass transfer coefficient. Additionally, a dimensionless reaction-convection number, NRC, which is a function of the power input per unit volume, is proposed, which has been appropriately employed in correlating the effectiveness factor for the types of processes in which convection may be the key resistance factor. © 1996 John Wiley & Sons, Inc.
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  • 70
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    Biotechnology and Bioengineering 51 (1996), S. 229-236 
    ISSN: 0006-3592
    Keywords: bioreactor ; fluidized bed ; murine granulocyte-macrophage colony stimulating factor ; Saccharomyces cerevisiae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) by immobilized yeast cells, Saccharomyces cerevisiae strain XV2181 (a/a, Trp1) containing plasmid pαADH2, in a fluidized bed bioreactor was studied at a 0.03 h-1 dilution rate and various particle loading rates ranging from 5% to 33% (v/v). Cells were immobilized on porous glass beads fluidized in an air-lift draft tube bioreactor. A selective medium containing glucose was used to start up the reactor. After reaching a stable cell concentration, the reactor feed was switched to a rich, nonselective medium containing ethanol as the carbon source for GM-CSF production. GM-CSF production increased initially and then dropped gradually to a stable level. During the same period, the fraction of plasmid-carrying cells declined continuously to a lower level, depending on the particle loading. The relatively stable GM-CSF production, despite the large decline in the fraction of plasmid-carrying cells, was attributed to cell immobilization. As the particle loading rate increased, the plasmid stability also increased. Also, as the particle loading increased from 5% to 33%, total cell density in the bioreactor increased from 16 to 36 g/L, and reactor volumetric productivity increased from 0.36 to 1.31 mg/L·h. However, the specific productivity of plasmid-carrying cells decreased from 0.55 to 0.07 mg/L·g cell. The decreased specific productivity at higher particle loading rates was attributed to reduced growth efficiency caused by nutrient limitations at higher cell densities. Both the reactor productivity and specific cell productivity increased by two- to threefold or higher when the dilution rate was increased from 0.03 to 0.07 h-1. © 1996 John Wiley & Sons, Inc.
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  • 71
    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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  • 72
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    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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  • 73
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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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  • 74
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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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  • 75
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    Biotechnology and Bioengineering 49 (1996), S. 601-610 
    ISSN: 0006-3592
    Keywords: aerobic ; anaerobic ; biomass separation ; bioreactor ; bubbleless ; oxygen mass transfer ; extraction of organic pollutants ; membrane ; wastewaters ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Combining membrane technology with biological reactors for the treatment of municipal and industrial wastewaters has led to the development of three generic membrane processes within bioreactors: for separation and recycle of solids; for bubbleless aeration of the bioreactor; and for extraction of priority organic pollutants from hostile industrial wastewaters. Commercial aerobic and anaerobic membrane separation bioreactors already provide a small footprint alternative to conventional biological treatment methods, producing a high-quality effluent at high organic loading rates. Both the bubbleless aeration and extractive membrane bioreactors are in the development stages. The former uses gas-permeable membranes to improve the mass transfer of oxygen to the bioreactor by providing bubbleless oxygen. By using a silicone membrane process, extractive membrane bioreactors transfer organic pollutants from chemically hostile wastewaters to a nutrient medium for subsequent biodegradation. All three membrane bioreactor (MBR) processes are comparatively and critically reviewed. © 1996 John Wiley & Sons, Inc.
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  • 76
    ISSN: 0006-3592
    Keywords: algal culture ; bioreactor ; bioregenerative system ; energy economy ; light-emitting diode (LED) ; microsecond pulse modulation ; Chlorella pyrenoidosa ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Light-emitting diodes (LEDs) were used as the sole light source in continuous culture of the green alga Chlorella pyrenoidosa. The LEDs applied show a peak emission at 659 nm with a half-power bandwidth of 30 nm. Selection of this wavelength range, which is optimal for excitation of chlorophylls a and b in their “red” absorption bands makes all photons emitted potentially suitable for photosynthesis. No need for additional supply of blue light was found. A standardized panel with 2 LEDs cm-2 fully covered one side of the culture vessel. At standard voltage in continuous operation the light output of the diode panel appeared more than sufficient to reach maximal growth. Flash operation (5-μs pulse duration) enables potential use of higher operating voltages which may render up to three times more light output. Flat airlift fermentor-type continuous culture devices were used to estimate steady state growth rates of Chlorella pyrenoidosa as a function of the light flux (μmol photons · m-2 · s-1) and the flashing frequency of the light-emitting diodes (which determines the duration of the dark “off” time between the 5-μs “on” pulses). At the fixed voltage and turbidostat setting applied a 20-kHz frequency, which equals dark periods of 45 μs, still permitted the maximum growth rate to become nearly reached. Lower frequencies fell short of sustaining the maximal growth rate. However, the light flux decrease resulting from lowering of the flash frequency appeared to reduce the observed growth rates less than in the case of a similar flux decrease with light originating from LEDs in continuous operation. Flash application also showed reduction of the quantum requirement for oxygen evolution at defined frequencies. The frequency domain of interest was between 2 and 14 kHz. LEDs may open interesting new perspectives for studies on optimization of mixing in mass algal culture via the possibility of separation of interests in the role of modulation on light energy conversion and saturation of nutrient supply. Use of flashing LEDs in indoor algal culture yielded a major gain in energy economy in comparison to luminescent light sources. © 1996 John Wiley & Sons, Inc.
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  • 77
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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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  • 78
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    Biotechnology and Bioengineering 57 (1998), S. 471-476 
    ISSN: 0006-3592
    Keywords: soil immobilization ; soil pollutants ; bioremediation ; bioreactor ; biofilm ; pentachlorophenol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new concept for the development of microbial consortia for the degradation of persistent soil pollutants and for pollutant treatment is proposed. The concept defined as “soil immobilization” is based on the entrapment of soil particles, showing microbial activity in degrading the target pollutant, into a solid membrane with a large pore size distribution. The particular hydrodynamic and mass transfer properties of this system result in a very efficient process. A new type of bioreactor is proposed for carrying out the immobilized soil process. The performance of the system was tested by developing a microbial system for the mineralization of pentachlorophenol (PCP). The results show that the volumetric efficiency of the process for PCP mineralization in the immobilized soil bioreactor is 1-3 orders of magnitude higher than reported literature values. Chlorine and carbon atoms of PCP are both nearly completely (99%) mineralized. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 471-476, 1998.
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  • 79
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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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  • 80
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    Biotechnology and Bioengineering 52 (1996), S. 507-517 
    ISSN: 0006-3592
    Keywords: oxygen mass transfer coefficient ; molecular weight ; polysaccharide fermentation ; bioreactor ; reciprocating plate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In most polysaccharide fermentations, the nature of the fermentation broth changes drastically with time and, as a result, the overall oxygen mass transfer coefficient (KLa) can vary by orders of magnitude. To obtain a better understanding of this phenomenon, an experimental program was devised to study the respective influence of molecular weight and concentration of dextran solutions on KLa. Experiments were conducted in a reciprocating plate bioreactor. This bioreactor uses a stack of perforated plates that is reciprocated axially in the column and it is therefore well suited for mixing viscous liquid broths and providing uniform overall mass transfer coefficients. The variation of KLa with the power input per unit volume and the superficial gas velocity were obtained for three ranges of molecular weights and five concentrations of dextran. In every medium, two regimes of operation were observed as a function of the power input per unit volume: a first regime, at low power inputs per unit volume where KLa remains constant until a threshold of power input is attained; and a second regime, which is characterized by a steep increase of KLa as a function of the power input per unit volume. The presence of dissolved biological macromolecules, not only because of their effect on the rheology of the medium but also because their effect on the gas-liquid interface, has a significant impact on KLa. It was found that, generally, small concentrations of polysaccharide favor oxygen mass transfer despite the increase in medium viscosity. However, the respective influence of polysaccharide concentration and molecular weight was different for the two regimes of operation. © 1996 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 54 (1997), S. 115-121 
    ISSN: 0006-3592
    Keywords: Methylobacterium organophilum ; high viscosity polysaccharide ; methylan ; multidisk mixer ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of shear stress on the production of a high viscosity polysaccharide, methylan, from methanol by Methylobacterium organophilum was investigated by using a multidisk mixer. It was observed in the multidisk mixer with defined shear stresses that the specific production rate of methylan increased gradually with increasing shear stress up to 30 Pa, and the production rate was constant beyond 30 Pa. This result suggested that the limited mass transfer from the medium into cells reduced methylan production. A novel bioreactor that provided the large volume of a high shear region was used to increase methylan production. Fed-batch cultures in the novel bioreactor were performed by the dissolved oxygen-stat method of methanol. When 1.13 g/L ammonium ion was added, the concentrations of cells of methylan were 31 and 20.6 g/L, respectively. The productions of cells and methylan in our designed bioreactor were 20 and 50% higher than those obtained in a conventional fermentor. The methylan content reached a maximum of 20.7 g/L in the bioreactor and the viscosity of the fermentation broth was 127 Pa · s, which corresponds to 68 g/L as a xanthan. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 115-121, 1997.
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  • 82
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 83
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 679-683 
    ISSN: 0006-3592
    Keywords: cultured epidermal autografts ; bioreactor ; keratinocyte cultures ; tissue engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this work was to engineer an automated system for the production of cultured epidermal autografts and sub-confluent cultured epidermal autografts. Human epidermal cells were grown directly on a transparent FEP film, which was held in place and surrounded by a polycarbonate growth chamber. The growth chambers were stacked to accommodate various surface area requirements. To monitor the development of the grafts, the upper-most growth chamber in the stack was periodically placed on a standard phase contrast microscope. The growth chambers were connected to a multi-channel peristaltic pump, which was controlled automatically to manage fluid-handling operations. Sub-confluent graft production involved removing the epidermal-film composite from the growth chambers and cutting desired graft geometries. Producing cultured epidermal autografts involved (1) removing the confluent epidermal-film composite from the growth chambers, (2) treating the composites with dispase, and (3) clipping the detached cultured epidermis to a synthetic support. Twelve to fifteen days were required to produce sub-confluent grafts (total surface area 3500-4500 cm2 50% confluent) and 18 to 24 d were required to produce standard cultured epidermal autografts (total surface area 3500-4500 cm2). The system reduces the tedious manual labor associated with producing cultured epidermal autografts. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:679-683, 1998.
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  • 84
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 633-641 
    ISSN: 0006-3592
    Keywords: bioreactor ; elastic sparger ; bubble size ; oxygen transfer efficiency ; characteristic mixing time ; wrinkled bubbles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Gas transfer and mixing were characterized in a 32-L bubble column reactor equipped with a commercially available rubber membrane diffuser. The performance of the membrane diffuser indicates that the slits in the membrane are best described as holes with elastic lids, acting as valves cutting off bubbles from the gas stream. The membrane diffuser thus functions as a one-way valve preventing backflow of liquid. Our design of the bottom plate of the reactor enabled us to optimize the aeration by changing the tension of the membrane. We thereby achieved mass transfer coefficients higher than those previously reported in bubble columns. A strong dependence of mass transfer on gas holdup and bubble size was indicated by estimates based on these two variables. The microalga, Rhodomonas sp., sensitive to chemical and physical stress, was maintained for 8 months in continuous culture with a productivity identical to cultures grown in stirred tank reactors. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 633-641, 1998.
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  • 85
    Electronic Resource
    Electronic Resource
    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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  • 86
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; metabolic oscillation ; Synchronization affector ; continuous culture ; Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: When the yeast Saccharomyces cerevisiae was grown under aerobic continuous culture, an autonomous shortperiod-sustained oscillation appeared. This oscillation was observed in concentrations of various extracellular and intracellular parameters, such as ethanol, acetate, glycogen, dissolved oxygen and intracellular pH. In this work the synchronization affecter of this oscillation was investigated. Ethanol was found not to be the synchronizer of the oscillation because a pulse of ethanol did not affect the phase or period of the oscillation. The oscillation was dependent on the aeration rate, i.e., the oscillation occurred only between 150 and 600 ml min-1. However, the oxygen concentration did not influence synchronization as an upward shift in the oxygen concentration of the gas flow did not affect the sustainability of the oscillation. On the other hand, synchronization was stopped by an enhanced gas flow rate, keeping dissolved oxygen tension at the oscillatory condition, suggesting that synchronization was caused by a volatile compound in the culture. A stepwise increase in carbon dioxide concentration of the gas flow rate ceased synchronization, yet the oscillation seem to continue in each individual cell. Oscillatory behaviour of intracellular pH and carbon dioxide evolution rate showed a phase difference of 90 degrees. Based on these facts it is postulated that carbon dioxide, through the influence of its dissociation on intracellular pH, could be the synchronization affector of the autonomous short-period-sustained oscillation of S. cerevisiae.
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