Appl Environ Microbiol, 1992 Mar, 58(3), 953 - 60 Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene; Tsien HC et al.; Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE) . The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned . It contained a 2.2-kb EcoRI fragment . With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO . Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis . Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE . The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes . Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe . When grown in media with limited copper, all of these bacteria degraded TCE . All of them are type II methanotrophs . The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M . trichosporium OB3b soluble MMO component B gene probe, although M . capsulatus Bath also produces a soluble MMO. Biotechniques, 1992 Feb, 12(2), 258 - 63 Use of perfluorocarbon emulsions in cell culture; Ju LK et al.; Perfluorocarbon emulsions were applied to hybridoma cultures grown in tissue culture tubes and column bioreactors . The oxygen transfer enhancement effect of perfluorocarbon emulsions was clearly demonstrated by the higher cell densities obtained in emulsion-supplemented systems . In addition, perfluorocarbon emulsions were shown to provide better cell suspension in a low-shear environment . The study in column bioreactors also suggested a cell protective effect of the employed perfluorocarbon emulsions in reducing the damage to cells by gas bubbles. Int J Artif Organs, 1992 Feb, 15(2), 126 - 30 Membrane bioreactors as hybrid artificial pancreas: experimental evaluation; Lombardi CP et al.; Results of cultured islet transplantation in the management of insulin-dependent diabetes are still unsatisfactory . The main problem preventing success is the swift and resolute host immune rejection . To obviate this we designed and experimented a model of bioartificial pancreas, made of polymeric hollow fibers, put into the blood circulation as an artero-venous bypass to immunoisolate endocrine tissue from leucocytes and immunoglobulins . We tested four different membrane bioreactors (BR1-4) . BR1 and 2 had seven hollow fibers, the others more than 6,000 smaller fibers . In BR4 a connecting tube with a high-permeability membrane was inserted between the islet compartment and the bioreactor outlet to improve the ultrafiltration flow . In vitro, the islets inside the bioreactor perfused with glucose solutions (300 mg%) showed a rapid, high insulin secretory response, related to the glucose stimulation . The use of the outside connection allowed a twofold increase of insulin production. Protein Expr Purif, 1992 Feb, 3(1), 27 - 35 Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture; Levin W et al.; Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor . The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days . Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2) . The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins . However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml . With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta. Appl Microbiol Biotechnol, 1992 Feb, 36(5), 611 - 7 Growth characteristics of Sanguinaria canadensis L . cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids; Rho D et al.; Sanguinaria canadensis L . plants were harvested from a local forest and calli were initiated from leaf explants . The production of benzophenanthridine alkaloids (i.e . sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S . canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes . Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%, g g-1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g-1) . Sanguinarine, chelirubine and chelerythrine were the only known alkaloids detected in cultured S . canadensis cells . Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion . The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor . The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days . The maximum sugar and nitrate consumption rates were 1.8 g l-1 per day and 2.3 mM per day respectively . The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O2 l-1 h-1 and 0.95 mmol CO2 l-1 h-1 respectively . The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g-1 dw) by day 15. J Biotechnol, 1992 Feb, 22(3), 311 - 27 Cultivation of animal cells in a reticulated vitreous carbon foam; Kent BL et al.; A reticulated vitreous carbon foam (RVCF) was used as a surface to cultivate a model anchorage-dependent animal cell line, 3T6 (mouse embryo fibroblast) . This fixed-surface bioreactor provided a low-shear, chemically-inert, and reusable environment for cell growth . An external medium recirculation loop allowed aeration, nutrient monitoring, and medium replacement without disturbing the cells . Optimal flow rates for the attachment and growth phases were determined . Growth rates comparable to static (T-flask and petri dish) cultures and agitated microcarrier cultures were achieved with appropriately high medium recirculation rates . Metabolic parameters were shown to be useful indicators of cell mass, although specific glucose consumption rates were considerably higher for cultures in the RVCF reactor . Oxygen supply was shown to be the most likely limiting factor for scaleup. J Biotechnol, 1992 Feb, 22(3), 291 - 8 Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors; van Lier FL et al.; Insect cells were exploited to produce bacterial beta-galactosidase by infecting them with a recombinant nuclear polyhedrosis virus (baculovirus) of Autographa californica . The insect cells were cultured in a continuous stirred tank reactor (CSTR) and led to a second CSTR where they were infected with a recombinant virus in which the lacZ gene from Escherichia coli was inserted . In the effluent of the production reactor, maximum activities of 15 units beta-galactosidase per 10(6) cells were measured . For about 25 d beta-galactosidase production remained constant, but then rapidly declined . This drop was due to a decrease in production of active beta-galactosidase rather than to inactivation of this enzyme . It was concluded that the reduced production was due to reduced polyhedrin promoter-driven synthesis. J Biotechnol, 1992 Feb, 22(3), 245 - 70 Further studies of the culture of mouse hybridomas in an agitated bioreactor with and without continuous sparging; Oh SK et al.; TB/C3 mouse hybridoma cells have been grown at 2 controlled dO2 conditions by headspace and sparged oxygenation . Also a variety of sparging rates and sparger sizes and positions have been employed . Headspace oxygenation at dO2 levels from 5% to 100% of saturation give essentially the same performance as controls . Sparging is generally damaging to cells, the extent of damage decreasing with reduced sparging rate until at below about 0.02 vvm results equivalent to the unsparged conditions are obtained . Damage is clearly linked with bubble-cell interactions at the air-medium interface where bubbles bursting in clusters and of a size less than 5 mm appear to be the most lethal . When the interaction of air sparging with the agitator flow leads to an increase in the number of smaller bubbles and cluster bursts, cell damage is further increased . Pluronic F-68 reduces damage very significantly . Biological aspects are briefly discussed in the light of various biological tests . The practical implications of this work for large scale, free suspension cell culture are outlined. In Vitro Cell Dev Biol, 1992 Jan, 28A(1), 47 - 60 Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels; Goodwin TJ et al.; A new low shear stress microcarrier culture system has been developed at NASA's Johnson Space Center that permits three-dimensional tissue culture . Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs) . RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system . Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer . Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation . In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered . The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters . The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development . Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices . Necrosis was minimal throughout the tissue masses . These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue. Adv Biochem Eng Biotechnol, 1992, 46, 187 - 223 Modelling the growth of filamentous fungi; Nielsen J; Despite the considerable industrial importance of filamentous fungi there have been very few attempts to model the complex growth process of these microorganisms . With a new generation of high performance, computerized bioreactors and new analytical techniques it is possible to obtain the necessary experimental data for setting up reliable structured models describing the growth process of filamentous fungi . It is therefore interesting to review the mathematical models described previously in the literature and the experimental data on which these models are built . Only structured models are considered due to the complex metabolism of filamentous fungi and to the natural cellular structuring of the biomass, i.e . the biomass can be divided into different cell types . In order to set up good structured models it is strictly necessary to have a detailed knowledge of the mechanisms underlying the growth process . This involves both biochemical insight and understanding of the interactions between different macromolecules and cytological organelles. Folia Microbiol (Praha), 1992, 37(2), 133 - 9 Phenomenological theory of substrate-induced acidification with application to Candida utilis dissimilating ethanol; Votruba J et al.; The mathematical theory of substrate-induced acidification by microorganisms was based on special formulation of mass and charge conservation laws for distinct species . The strength of the theory was tested on the ethanol-induced acidification by Candida utilis in pure water and in phthalate buffer . We showed that the theory may be used to approximate, extrapolate and predict the course of apparent acidification including the effects of buffering capacity of the broth . It makes it possible to formulate mathematical models that are suitable for process simulation and computer control of bioreactors when pH is used as a state variable. Vaccine, 1992, 10(13), 896 - 9 Contribution to rabies prevention; Sureau P; After the end of the Second World War, an outbreak of fox rabies invaded Europe . For the immunization of human populations and domestic animals against the risk of rabies transmitted by infected wild animals, it appeared necessary to replace the first generation of rabies vaccines (nerve tissue vaccines) by more potent and safer vaccines . The European vaccine manufacturers, in close collaboration with the research institutes engaged in rabies research, soon and quickly developed a second generation of rabies vaccines, produced in cell cultures including continuous cell lines grown in bioreactors of industrial scale . The third generation of rabies vaccines is already available: the vaccinia-rabies glycoprotein recombinant vaccine is presently applied on a large scale in some European countries for immunization of wildlife . The canarypox recombinant vaccine has already been considered and successfully tested for human immunization. Biomater Artif Cells Immobilization Biotechnol, 1992, 20(5), 1177 - 92 Hybrid artificial pancreas: islet transplantation inside membrane bioreactors; Lombardi CP et al.; The use of pancreatic islet transplantation in membrane bioreactors put in vascular circuits aims at resetting the glucose homeostasis in diabetic or pancreatectomized patients, avoiding immune host rejection . Our experience was carried out at following stages: porcine pancreas explantation and enzymatic separation of endocrine tissue from exocrine fraction by collagenase; evaluation of islet functionality (culture tests); in vitro tests of the islets-bioreactor system, to assess the metabolic response to the glucose; in vivo evaluation to assay the haemodynamic behaviour . The trials showed a good metabolic bioreactor functionality and a decreasing incidence of coagulative problems. Res Exp Med (Berl), 1992, 192(5), 305 - 16 Bioartificial endocrine pancreas: foreign-body reaction and effectiveness of diffusional transport of insulin and oxygen after long-term implantation of hollow fibers into rats; Bodziony J; Foreign body reaction of the liver, kidney, and subcutaneous tissue of rats after implantation of hollow fibers (HF) for 1, 3, 6, and 12 months and its influence on the effectiveness of diffusional transport of insulin and oxygen were investigated . The highest degree of fibrosis was observed after subcutaneous implantation of HF and the lowest degree after implantation into the kidney . Histochemical staining of the fibrous capsule showed a tissue-dependent domination of the collagenous fibrils . After 90 min of perfusion 33% of the insulin contained in HF diffused out of the nonimplanted fiber, after 120 min, 73% and after 180 min, 100% . Hollow fibers, which were removed with a surrounding connective capsule after an implantation period of 1 year, showed even better permeability for insulin than nonimplanted HF . The tension of oxygen in the lumen of the implanted hollow fibers was 42 mm Hg after implantation into the kidney and 30 mmHg after implantation into the liver . The oxygen present inside an HF that is implanted into a kidney and liver was consumed by 100 islets in 3.9 and 2.8 min, respectively . It was concluded that to achieve acceptable results in the construction of bioartificial pancreas more research activities should be performed on the diffusion and consumption of oxygen in the bioreactor. Cytotechnology, 1992, 9(1-3), 59 - 67 Optimal medium use for continuous high density perfusion processes; Buntemeyer H et al.; For maintenance of high cell density in continuous perfusion processes not only feeding with substrates but also removal of inhibitors and toxic waste products are of special interest . High perfusion rates cause large volumes of product containing medium which have to be processed in product isolation . In order to minimize these volumes concentrated feed solutions of optimized medium are used . On the other hand, such media may cause high concentrations of toxic or inhibitory metabolites which can negatively influence cell growth and product formation . Especially, if the spent medium (or special parts of it) is used again after product isolation, the removal or even better the control of inhibitor production is of highest importance . We have developed a continuous fermentation concept and system (continuous medium cycle bioreactor, cMCB) in which both limitation and inhibition effects can be generated to identify special substances as limiting or inhibitory components . With the results from those experiments it was possible to lower the total perfusion rate during serum-free perfusion cultures of hybridoma cells and to obtain an optimal substrate utilization . The advantages for decreasing the production costs (for media, special supplements and product isolation) are obvious . The other aim of this study was to identify secreted metabolic waste products as inhibitor or toxic metabolite. Cytotechnology, 1992, 9(1-3), 51 - 7 The membrane dialysis bioreactor with integrated radial-flow fixed bed--a new approach for continuous cultivation of animal cells; Bohmann A et al.; A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran carriers over a period of 6 weeks . Antibodies accumulated to an average of 100 mg l-1, approx . 10 times more than in fixed bed cultures without dialysis membrane . Serum costs could be reduced about 85% due to an appropriate feeding strategy . Siran carriers with 3-5 mm diameter showed an advantage compared to those with 1-2 mm diameter . For the 3-5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s-1 and 0.75 mm s-1 . At higher velocities cells are washed out of the bed . Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days . From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected. Cytotechnology, 1992, 9(1-3), 41 - 9 Modified CelliGen-packed bed bioreactors for hybridoma cell cultures; Wang G et al.; This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG1 monoclonal antibody . The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column . In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen bioreactor . In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller . Both configurations are packed with disk carriers made from a non-woven polyester fabric . During the steady-state phase of continuous operation, a cell density of 10(8) cells per cm3 of bed volume was obtained in both bioreactor configurations . The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1%) of serum as well as serum-free media . These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature . The media flows evenly over the cells and produces very low shear forces . These systems are easy to set up and operate for prolonged periods of time . The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed. Cytotechnology, 1992, 9(1-3), 3 - 9 Maximisation of perfusion systems and process comparison with batch-type cultures . Maximisation of perfusion cultures; Griffiths JB et al.; A comparison of cell yields and monoclonal antibody productivity from the same hybridoma has been made in a wide range of cell bioreactors including both batch and continuous perfusion cultures . The most productive systems were based on porous microcarriers in fixed and fluidised beds which can be operated with a high degree of efficiency and reliability from the physico-chemical engineering point of view . Further improvements should be possible by improving the physiological environment in dense cell cultures, as indicated by the preliminary studies that are described . These include experimental data showing the relationship between monoclonal antibody production rates with glucose, glutamine, ammonia, and oxygen levels in microporous beads . The results strongly indicate that perfusion processes that are scaleable in both volume and cell density can significantly reduce production costs . Manufacturers of biologicals from animal cells now have a choice between the proven batch-type processes and reliable perfusion systems based on microporous beads. Cytotechnology, 1992, 9(1-3), 29 - 40 Design and performance of a trickle-bed bioreactor with immobilized hybridoma cells; Phillips HA et al.; A trickle-bed system employing inert matrices of vermiculite or polyurethane foam packed in the downcomer section of a split-flow air-lift reactor has been developed for hybridoma culture to enhance antibody productivity . This quiescent condition favoured occlusion and allowed the cells to achieve densities twelve fold greater (12.8 x 10(6) cells/ml reactor for polyurethane foam) than in free cell suspension . The reactor was operated in a cyclic batch mode whereby defined volumes of medium were periodically withdrawn and replaced with equal volumes of fresh medium . The pH of the medium was used as the indicator of the feeding schedule . Glucose, lactate and ammonia concentrations reached a stationary value after 5 days . With vermiculite packing, a monoclonal antibody (MAb) concentration of 2.4 mg/l was achieved after 12 days . The MAb concentration declined then increased to a value of 1.8 mg/l . In the polyurethane foam average monoclonal antibody (MAb) concentrations reached a stationary value of 1.1 mg/l in the first 20 days and increased to a new stationary state value of 2.1 mg/l for the remainder of the production . MAb productivity in the trickle-bed reactor was 0.3 mg/l.d (polyurethane foam) and 0.18 mg/l.d (vermiculite) in comparison to 0.12 mg/l.d for free cell suspension . This trickle-bed system seems to be an attractive way of increasing MAb productivity in culture. Cytotechnology, 1992, 9(1-3), 141 - 7 Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus; Wu J et al.; Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's) . In temperature-shift experiments (cell growth at 33 degrees C followed by virus replication at 27 degrees C 3-4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested . In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02) . This result is contrary to that obtained in constant-temperature culture (27 degrees C for both cell growth and virus replication) . Virus and CAT production was greatly improved when the entire culture was run at constant temperature . It appeared that infected cells were severely damaged at 33 degrees C (6 degrees C above the optimal 27 degrees C), resulting in little or no virus and protein production . As a result of these temperature-shift experiments, a larger-scale (14 1 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27 degrees C) to produce recombinant protein (beta-galactosidase) . A cell density as high as 1 x 10(7) cells.ml-1, and a beta-gal concentration of up to 104,000 unit.ml-1 were achieved. Cytotechnology, 1992, 9(1-3), 11 - 9 An engineering analysis of rotating sieves for hybridoma cell retention in stirred tank bioreactors; Favre E et al.; The use of internal rotating sieves for perfused hybridoma culture offers unique advantages but has been up to now largely empirical . Calculations have been performed on a 15 l spinfilter stirred tank in order to have an idea of hydrodynamic conditions inside and outside the rotating sieve . The large peripheral velocity value, resulting from sieve rotation (compared to axial and radial velocities) is expected to affect strongly sieve surface colonization by cells; this is confirmed by lab scale experiments, showing that cell colonization is prevented providing sieve rotation exceeds a defined value (around 0.6 m.s.1 tip speed); the fluid removal force calculated under these conditions appears to be in the range of 10 pN, similar to the adhesion force already reported for mammalian cells attached to inorganic substrata. Cytotechnology, 1992, 8(3), 207 - 14 Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry; Borth N et al.; As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors . The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line . This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary . Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture. Cytotechnology, 1992, 8(3), 179 - 87 A comparison of simple growth vessels and a specially designed bioreactor for the cultivation of hybridoma cells; Persson B et al.; Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture conditions . The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large differences in vessel and agitator geometry and aeration mode . Furthermore, some data indicate that the hydrodynamic stress level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the reactors. Cytotechnology, 1992, 8(1), 85 - 8 High density culture of HeLa cells in a CelliGen perfusion system; Chen Y et al.; A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor . Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems . This system successfully propagated HeLa cells to over 11 million viable cells per milliliter . Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate . Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells . Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor. Appl Microbiol Biotechnol, 1992 Jan, 36(4), 446 - 51 A model for penicillin production with and without temperature shift after the growth phase; Kluge M et al.; A strain of Penicillium chrysogenum producing about 8 milligrams/l of penicillin V, was cultivated in a 10-1 bioreactor . Under carbon (C)-limitation during the production phase a glucose/ammonium sulphate mixture was fed using microprocessor control . When the temperature was shifted from 25 degrees C to 30 degrees C at the end of the active growth phase, the specific penicillin production rate was increased by 30%, while the yield remained constant . Maximal productivity without sporulation was obtained when the net growth rate of the active (respiring and producing) biomass, estimated by measuring the respiration rate under defined conditions, was equal to or higher than 0.004 h-1 . A model was developed for penicillin fermentation during C-limitation possessing the following properties: (1) the model is based on ordinary differential equations; (2) the influence of different nutrients is considered; (3) the model recognizes two cell types (active and inactive); (4) the model describes the influence of a temperature shift at the end of the vigorous growth phase. J Biotechnol, 1992 Jan, 22(1-2), 51 - 68 Safety and economic aspects of continuous mammalian cell culture; Werner RG et al.; Mammalian cell cultures are the most appropriate host cells for recombinant DNA derived products if complex protein structures have to be synthesized in their native form . Due to their physiological behaviour they grow either adherent or in suspension . For the attachment of adherent cells, microcarriers or wire springs can be applied to increase the internal surface of the bioreactor . Both systems provide a simplified media exchange but, however, show some limitations in scale up . In contrast, suspension culture systems as homogeneous systems independent of any carrier have not shown any limitation in scale up . Because most cell lines which are of commercial interest grow in suspension, this technology is best advanced and used in batch and continuous mode . Although mammalian cell cultures are sensitive to hydrodynamic shear forces, technologies for deep tank production are developed which allow stirrer tip speed of up to 1.5 m s-1 sufficient for oxygen uptake, suspension of cells and homogeneous supply with nutrients . For long term bioprocesses without selection pressure it has to be considered that transformed cell lines might show genetic instability due to their variations of chromosomes . In addition, sterile technology becomes an important factor in long term bioprocesses . The decision as to which cell culture system should be chosen, whether batch or continuous processes should be applied essentially is based on the capital investment, the amount of material to be produced, genetic stability of the production cell line, reliability of sterile technology and the flexibility required in the production plant . Under the assumption that 20 kg of a protein have to be produced per year and the same product concentrations in the harvest fluid are reached in the batch process and for instance in the chemostat, it can be considered that the capital investment for one 10,000 l batch process and a 2 x 2,000 l continuous process, necessary to produce the amount of material, is comparable . Risk of microbial contamination or technical failure can be considered to be fairly low in the batch process . The economic risk for long term bioprocess in the chemostat can be expected to be medium and high in the perfusion system which is in the large scale not technically fully satisfactory . In addition, due to the longer down time period after contaminations and the start up of the continuous process, the annual yield of the batch process can be considered to be higher.(ABSTRACT TRUNCATED AT 400 WORDS) J Biotechnol, 1992 Jan, 22(1-2), 41 - 50 Effect of specific growth rates on productivity in continuous open and partial cell retention animal cell bioreactors; Robinson DK et al.; A clonal derivative of a transfectant of the SP2/0 myeloma cell line producing a chimeric monoclonal antibody was cultivated in both continuous open and continuous partially-closed bioreactors . Using an open system for the determination of kinetic parameters, we showed that the production of this chimeric mAb was growth associated . As such, the volumetric productivity increased linearly with increasing dilution rate up to the maximum dilution rate . Three continuous cultivations employing partial cell retention were conducted . In agreement with mathematical predictions, the product titer and volumetric productivity were independent of the degree of cell retention when the total dilution was held constant . When cells were maintained at a low specific growth rate, the product titer was independent of dilution rate and the volumetric productivity increased with increasing dilution rate, again in agreement with mathematical predictions . Since the partially-closed bioreactor could be operated at dilution rates in excess of the maximum specific cellular growth rate, volumetric productivities were greater than those achievable in the open bioreactor . However, when cells were maintained at a high specific growth rate, cell accumulation was limited and product titers decreased at high dilution rates . Therefore, the volumetric productivity in this latter case did not increase at higher dilution rates. J Biotechnol, 1992 Jan, 22(1-2), 31 - 40 Continuous hybridoma suspension cultures with and without cell retention: kinetics of growth, metabolism and product formation; Schmid G et al.; A laboratory scale bioreactor was constructed from glass and polycarbonate materials whereby a track-etch membrane (3 microns pore diameter) was integrated into its two-part bottom flange . The reactor performance was evaluated for continuous hybridoma suspension cultures under various conditions of cell retention . A total retention experiment demonstrated that this type of stirred tank reactor cannot be operated at near zero growth rate conditions . Instead, at steady viable cell concentrations of congruent to 3 x 10(6) cells per ml, specific growth and death rates were estimated at 0.60 +/- 0.06 d-1 . Specific substrate (glucose, glutamine, O2, amino acids) consumption, by-product (ammonia, alanine, amino acids) and product (antibody) production rates as well as various apparent molar yield coefficients were obtained and are compared to metabolic quotients and yield coefficients previously calculated from standard continuous culture experiments, i.e., without cell retention, at specific growth rates of 0.63 and 1.24 d-1 . Furthermore, steady-state data on viable cell and antibody concentrations, spec . mAb productivities, and space-time yields determined before and after a step change (2.5-fold increase) in dilution rate at identical specific growth rates mu are presented. J Biotechnol, 1992 Jan, 22(1-2), 153 - 69 Heat shock gene expression in continuous cultures of Escherichia coli; Heitzer A et al.; Temperature inducible systems for the controlled expression of recombinant genes are finding increasing industrial applications . These involve either short or long term exposure of the process culture to superoptimum temperatures . It is well known that bacteria respond to a sudden increase in their environmental temperature with an immediate transient increase in the synthesis rates of specific heat shock proteins . The use of continuous flow processes for the production of recombinant proteins would allow higher productivity and smaller scale bioreactors . However, the induction patterns of heat shock proteins in continuous culture after defined heat shocks are not well defined despite a large amount of information which is now available concerning heat shock protein induction in batch cultures . An overview of this information is presented to enable a better understanding of the response in continuous cultures . The latter was investigated by monitoring the transient expression of a representative heat shock gene, htpG, in E . coli in continuous culture . The relative magnitude of the response was found to be both temperature and exposure time dependent, but growth rate independent . Changing medium composition resulted in both different steady and transient state expression levels. Enzyme Microb Technol, 1992 Jan, 14(1), 58 - 63 Controlling and predicting monoclonal antibody production in hollow-fiber bioreactors; Handa-Corrigan A et al.; A simple optimization strategy is described which enables monoclonal antibody (MCA) production in hollow-fiber bioreactors to be controlled and predicted . The MCA production rate is demonstrated to increase linearly with the uptake rates of glucose and glutamine and with the production rates of lactate and ammonia . The uptake and production rates of these metabolites can, in turn, be predicted from the pumping rates of basal medium to the bioreactor . We recommend a period of 2 weeks at the start of the cultivation when intensive assaying and monitoring should be carried out . After this period, the medium flow rate and MCA production rate may be predicted by linear extrapolation. Chirurgie, 1992, 118(10), 672 - 7 {Extracorporeal bio-artificial liver using isolated hepatocytes . An experimental study in the rat}; Fremond B et al.; A new type of bioartificial liver using isolated hepatocytes immobilized in alginate beads was developed and its capacity of correcting the metabolic deficiency of bilirubin conjugation in Gunn rats was assessed . Hepatocytes were isolated from Sprague-Dawley rats using the in situ collagenase perfusion technique, and they were then immobilized in Calcium alginate beads . The capacity of these immobilized hepatocytes to conjugate in vitro bilirubin was checked as compared to monolayer hepatocyte culture . The bioartificial liver consisted in a cylindrical bioreactor containing either alginate beads and hepatocytes (test group), or only alginate beads (control group) . Gunn rats were connected to this bioreactor through and extracorporeal circulation system, and "bile samples were collected" every hour . Bilirubin mono and biconjugates were dosed in the bile using the high-performance liquid chromatography technique . The viability of alginate immobilized hepatocytes, determined before and after each experiment, was stable at 75% . In the test group, the total conjugate concentration rapidly increased to reach a maximum value of 204 +/- 16 microns after 3 hours, while in the control group, there were only conjugate traces (1 micron) . These results show that the bioartificial liver is an efficient means to temporarily correct genetic deficiency in Gunn rats . Such a system could be of therapeutic interest in case of acute hepatic insufficiency. Asia Pac J Public Health, 1992-93, 6(2), 40 - 5 Environmental biotechnology: biotechnology solutions for a global environmental problem, hazardous chemical wastes; Omenn GS; Biotechnology has a growing place in the remediation of hazardous waste sites throughout the world, and especially in Asia where population density is high and land and fresh water are scarce . In-situ bioremediation has been demonstrated already to be highly effective for petroleum hydrocarbons (alkanes, aromatics, polychlorophenols) and organophosphate pesticides in soils and for gasoline by-products (benzene, toluene, xylene) and chlorinated solvents (trichloroethylene) in groundwater . Heavy metals and PCBs are not suitable for bioremediation . Environmental biotechnology includes solid-phase and slurry-phase bioremediation for contaminated soils and site-specific bioreactors for contaminated groundwater . Specific examples are presented . From a policy point of view, accumulated wastes must be detoxified, preferably at sites where they already exist . We cannot continue to rely on their removal and disposal "elsewhere" . For current waste streams, we must minimize the volumes and toxicity . Environmental biotechnology will play a key role. Chin J Biotechnol, 1992, 8(2), 123 - 30 Mass culture of anchorage-dependent animal cells with newly born calf skin collagen membrane and microcarrier; Chen Y et al.; It was shown that collagen substrate enhances the growth as well as the differentiation of many cells in culture . Collagen is one of the major fibrous proteins of animal bodies and the pure collagen used in this experiment was extracted from the newly born calf skin by chemical and biochemical methods . The analytical results obtained by ion-exchange chromatography and electrophoresis showed that the main components of denatured collagen were alpha monomers and beta dimers . The collagen and denatured collagen membranes were prepared by coating their solution on peteri dishes . Various types of cells were cultured on these membranes after being irradiated by ultraviolet ray . The denatured collagen was proved a good substratum for culturing anchorage dependent cells . A denatured collagen (gelatin) microcarrier, GT-2 was obtained by cross-linking gelatin with glutaraldehyde in a suspension polymerization process . These microcarriers were used successfully to culture various anchorage-dependent cells such as Vero, CHO, Bowes and fish cells in varying scales, including T-flasks, spinning bottles, revolving bottles and 1.5 l and 20 l bioreactors. Chin J Biotechnol, 1992, 8(3), 179 - 86 A perfusion system for high productivity of monoclonal antibody by hybridoma cells in a CelliGen bioreactor; Chen Y; DA4-4 (ATCC HB57) is a shear sensitive mouse-mouse hybridoma cell, producing an IgG1 monoclonal antibody against human IgM . It was grown successfully in a perfusion propagation system consisting of a 1.5 CelliGen stirred bioreactor and a hollow fiber cartridge . CelliGen system produced low shear force and cells could grow well at high agitation of 150 rpm . The culture was maintained for 40 days and cell number reached 20 x 10(6)/ml . Maximal monoclonal antibody concentration was 4.75 mg/ml . This system produced about 1.0 g of antibody every day at its peaks. Biosens Bioelectron, 1992, 7(9), 631 - 5 Implementation of a thermal biosensor in a process environment: on-line monitoring of penicillin V in production-scale fermentations; Rank M et al.; The production of penicillin V was monitored in 0.5 m3 and 160 m3 bioreactors . The thermal biosensor was an enzyme thermistor modified for split-flow analysis . The heat signal generated in the enzyme column was corrected for any nonspecific heat with the use of an identical but inactive reference column . The on-line monitoring was performed in the fermentation pilot plant and in a fermentation plant of Novo Nordisk A/S . Immobilized beta-lactamase was used to monitor three consecutive 0.5 m3 penicillin fermentations . Broth samples were continuously filtered through a tangential flow filtration unit in a sterile external loop . The on-line penicillin V values were 10% higher than those obtained by off-line HPLC analysis . Alternatively a polypropylene filtration probe was inserted into a 160 m3 bioreactor and samples were withdrawn at 0.5 ml/min . The same experiments were repeated with purified and immobilized penicillin V acylase . The on-line penicillin V values obtained with this enzyme correlated very well with those from HPLC analysis . The on-line monitoring was controlled and analysed by a software program written in Labtech Notebook. Immunol Res, 1992, 11(3-4), 181 - 90 Murine soluble Fc gamma receptors/IgG-binding factors (IgG-BF): analysis of the relation to Fc gamma RII and production of milligram quantities of biologically active recombinant IgG-BF; Sautes C et al.; The production of soluble forms of low-affinity Fc gamma R by cells expressing recombinant or natural membrane Fc gamma RII, and the structural relationships between these soluble receptors and membrane Fc gamma RII are described . We show that 37-40 kD soluble Fc gamma RII, corresponding to the two N-terminal domains of Fc gamma RII and binding to IgG, are spontaneously produced in vitro by cleavage of membrane Fc gamma RII . Moreover, we describe methods to produce and purify to homogeneity large quantities of endotoxin-free recombinant IgG-binding factor (rIgG-BF) from the culture medium of a cell line transfected with a mutated Fc gamma RII cDNA . These methods include the use of bioreactors for culturing transfected fibroblasts and the purification of rIgG-BF by ion-exchange chromatography and hydrophobic-interaction chromatography . By using such procedures, about 2.4 mg of rIgG-BF were purified from 1 liter of culture medium of transfected fibroblasts . Like natural IgG-BF, the 95-99% pure rIgG-BF suppressed, in a dose-dependent manner, secondary in vitro IgG antibody responses to sheep red blood cells. Ann N Y Acad Sci, 1991 Dec 27, 646, 322 - 33 Integration of cell culture with continuous, on-line sterile downstream processing; Grandics P et al.; The development of an integrated system for the continuous, automated production of pure cell culture derived proteins is discussed . The system comprises a cell culture subunit for the continuous culture of mammalian cells and a purification subunit linked on-line to the cell culture subunit . The cells are compartmentalized and continuously perfused with culture medium . The cell culture medium leaving the bioreactor is perfused through a sterile immunoaffinity column that instantaneously removes the product from the culture fluid . This results in improved product quality because the product is quickly removed from the cell culture, thus minimizing contact with degradative enzymes . The culture medium, stripped from the secreted product, is recirculated into the bioreactor . The system allows simple, automated, and economical production of purified proteins with higher quality than that possible with current production methods . The integration also allows on-line, real-time process monitoring, thus simplifying process development and allowing more consistent production of biologics. Ann N Y Acad Sci, 1991 Dec 27, 646, 300 - 6 High cell density fermentation of recombinant Escherichia coli with computer-controlled optimal growth rate; Knorre WA et al.; In recent years recombinant DNA technology has enabled us to produce various proteins of therapeutic importance with microorganisms . As an appropriate host organism, E . coli plays a dominant role . Yields of E . coli dry cell mass in shaker flask culture range from 1-2 g/L, whereas in fermentors up to 10 g dry cells/L can be achieved . ZIMET and GBF have developed a high cell density fermentation process that produces E . coli (on a glucose/mineral salt medium) up to more than 100 g dry cells/L in a special fed-batch mode . This cultivation strategy prevents oxygen limitation and hence the accumulation of acetate and other metabolic byproducts . The specific growth rate can be adjusted so that product formation reaches its optimum value . An example of the production of alpha1-interferon is presented . The high cell density fermentations were realized in 30- and 450-L Chemap fermentors (ZIMET) and in a three-stage bioreactor scale-up system (72, 300, and 1,500 L) developed in cooperation with GBF and B . Braun Melsungen AG . Multiloop controllers were used to control the process variables. Appl Biochem Biotechnol, 1991 Dec, 31(3), 283 - 310 Review and patents and literature . The use of insect cell cultures for recombinant protein synthesis: Engineering aspects; Murhammer DW; The use of the insect cell/baculovirus expression system for producing recombinant proteins of bacterial, plant, insect, and mammalian origin has become widespread . The popularity of this eukaryotic expression system is due to many factors, including (1) potentially high protein expression levels, (2) ease and speed of genetic engineering, (3) ability to accommodate large DNA inserts, (4) protein processing similar to higher eukaryotic cells (e.g., mammalia cells), and (5) ease of insect cell growth (e.g., suspension growth) . The following review of the literature discusses two engineering aspects of recombinant protein synthesis by insect cell cultures: bioreactor scale-up and insect cell line selection . Following this review patent abstracts and additional literature pertaining to expression of recombinant proteins in insect cell culture are listed. Biotechniques . 1991 Dec;11(6):702, 704, 706. Culture of eukaryotic cells with macro-reticulate buffers: fermentation of cellulolytic fungi; Pompei R et al.; Fermentation of fungi for large-scale production of extracellular cellulolytic enzymes requires a strict control of pH . At the lab scale, where bioreactors are not available, a culture in the exponential growth phase requires frequent manual pH adjustments . When fungi are grown in the presence of macroreticulate buffers, the culture is stable and does not require any pH control for as long as two weeks . These insoluble buffers are polyacrylamide beads (e.g., 10%T, 8%C) containing acrylamido weak acids and bases in such ratios as to unequivocally define a single pH value along the pH sale . At such pH, the macroreticulate buffers possess a strong buffering power (up to 100 milliequivalent liter-1 pH-1) . In the present example, a Trichoderma sp . strain is grown in the presence of 12% beads (v/v) with an isoelectric point of 5.6, containing 100 mM of a pK 6.2 weak acrylamido base and 89 mM of a pK 4.6 weak acrylamido acid . Enzyme production (exoglucanase, endoglucanase, xylanase, beta-glucosidase) is as good as (and often better than) the control in which the pH is adjusted manually 2-3 times/day. Artif Organs, 1991 Dec, 15(6), 474 - 80 Encapsulated multicellular spheroids of rat hepatocytes produce albumin and urea in a spouted bed circulating culture system; Takabatake H et al.; Multicellular spheroids are spherical cell-aggregates that retain tridimensional architecture and tissue-specific functions . For use of multicellular spheroids of hepatocytes in a bioreactor for hybrid artificial liver support, we studied the effect of encapsulation and circulating culture on their integrity and tissue-specific functions . Multicellular spheroids of rat hepatocytes were encapsulated into microdroplets of calcium alginate gel and were used as a bioreactor in medium circulating in a spouted bed chamber . Approximately 10% of the hepatocytes of an adult rat were entrapped in a bioreactor chamber, connected to a gas exchanger and a medium reservoir . The total bed volume of the system was 250 ml . The pH and DO2 of the hormonally defined circulating medium was maintained constantly . Albumin and urea were produced in a linear fashion for 64 h at the rates of 0.02 micrograms/microgram cell protein/day and 0.15-0.2 ng/micrograms cell protein/day, respectively . Viability and structural stability of the spheroids were well preserved after the culture period . These results indicate that these encapsulated multicellular hepatocyte spheroids will provide a useful bioreactor for the continuous production of albumin, in vitro and also a prototype hybrid artificial liver support. Appl Microbiol Biotechnol, 1991 Dec, 36(3), 324 - 6 High albumin production by multicellular spheroids of adult rat hepatocytes formed in the pores of polyurethane foam; Matsushita T et al.; Adult rat hepatocytes formed spherical multicellular aggregates (spheroids) when they were cultured in the pores of polyurethane foam (PUF) . The diameter of the spheroids was within the range 100-200 microns . These spheroids partly attached and immobilized in the PUF pores for at least 2 weeks . The albumin production rate by the spheroids increased up to 17.0 micrograms/10(6) nuclei per day during the first 6 days and maintained at a high level for 2 weeks . In contrast, the albumin production rate by the monolayer markedly decreased after 3 days . The spheroid culture using PUF seems to be a convenient and simple method for maintaining some differentiated functions of hepatocytes and for making a bioreactor using the function of spheroids. Trends Biotechnol, 1991 Dec, 9(12), 427 - 37 Fluid-mechanical damage of animal cells in bioreactors; Papoutsakis ET; The fluid-mechanical and some biological aspects of damage to animal cells in bioreactors due to agitation and/or aeration are attracting renewed attention . In microcarrier bioreactors, cell damage is due to forces generated by the interaction of microcarrier beads with each other and also with small turbulent eddies . For freely suspended cells grown in mixed bioreactors, cell damage is most frequently due to bubble breakup or fast-draining liquid films around rearranging gas-liquid interfaces. Appl Microbiol Biotechnol, 1991 Nov, 36(2), 208 - 10 A novel micromanipulation technique for measuring the bursting strength of single mammalian cells; Zhang Z et al.; Information about the bursting strength of animal cells is essential if the mechanisms of cell damage in bioreactors are to be understood, and if cell mechanical properties are ever to be related to cell structure and physiology . We have developed a novel cell compression technique that makes it possible to directly measure the bursting strength of single mammalian cells, and to infer information about cell mechanical properties. Enzyme Microb Technol, 1991 Nov, 13(11), 913 - 9 Biological responses of hybridoma cells to hydrodynamic shear in an agitated bioreactor; Abu-Reesh I et al.; Effects of long-term hydrodynamic shear on hybridoma cells were investigated in a 250-ml continuous stirred-tank reactor (CSTR) . Cells grown at steady state were subjected to step changes in agitation rates . Cell viability, glucose consumption, and monoclonal antibody (MAb) production were determined at high agitation rates and compared with the control (100 rev min-1) . Impeller tip speeds higher than 40 cm s-1 caused a significant drop in cell concentration and respiration activity, and increased lactate dehydrogenase (LDH) release to the culture medium . Also, high agitation speeds caused a decrease in MAb concentration and an increase in specific glucose consumption rate . The effects of dilution rate and serum concentration on the sensitivity of hybridoma cells to hydrodynamic shear were determined . Serum was found to protect the cells against shear damage and had a significant positive effect on hybridoma growth and MAb production . Shear damage on cells in CSTR was approximated to first-order kinetics . The death rate constant increased sharply at impeller tip speeds above 40 cm s-1. Enzyme Microb Technol, 1991 Nov, 13(11), 882 - 92 Large-scale (20 l) culture of surface-immobilized Catharanthus roseus cells; Archambault J; Surface-immobilized C . roseus cell cultures were grown in a 20-l modified airlift bioreactor operated at 0.51 vvm (kLa approximately 8 h-1) under various gassing regimes {air, 2% (v/v) and 5% CO2} . Extracellular ammonium, phosphate, and nitrate ions as well as carbohydrate uptake and pH value of the medium were monitored together with on-line dissolved oxygen concentration, conductivity of the medium, and carbon dioxide production rate (CPR) of the cultures . Cultures supplemented with 2% CO2 showed higher nitrate (5.0-7.0 mM d-1) and carbohydrate (3.3 g l-1 d-1) uptake rates and biomass production (mu approximately 0.24 d-1, yield approximately 0.33 g dw g CHO-1 and 7.4 g dw L-1) as compared to air (3.6 mM d-1, 2.1 g l-1 d-1; 0.20 d-1, 0.25 g dw g CHO-1 and 5 g dw l-1) and 5% CO2 (2.0-3.6 mM d-1, 2.0 g l-1 d-1; 0.11 d-1, 0.20 g dw g CHO-1 and 5 g dw l-1) cultures and as reported previously for suspension cultures . In addition, air and 5% CO2 cultures displayed incomplete carbohydrate uptake and, more important, phosphate and ammonium ion release into the medium at the end, which was ascribed to loss of viability . This was not observed for 2% CO2 immobilized bioreactor as well as shake flask control suspension cultures, which suggests that sparged C . roseus surface-immobilized cell cultures require 2% CO2 supplementation of the gas phase for both maximum growth and retained viability . The maximum CPRs of all cultures were in the same range (2.1-2.8 mM CO2 l-1 h-1) . However, the estimated maximum specific CO2 production rates of 2% CO2 and 5% CO2 immobilized cultures (0.6 mM g dw-1 h-1) were lower than those found for air-sparged immobilized cultures (1.0-1.3 mM g dw-1 h-1) . These rates are significantly higher than those reported in the literature for C . roseus cell suspension cultures performed in bioreactors gassed with air (approximately 0.2-0.55 mM g dw-1 h-1). Enzyme Microb Technol, 1991 Nov, 13(11), 873 - 81 Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing; Kasehagen C et al.; The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter {10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness} inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation . The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily . The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks . The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions . Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities . In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Prog, 1991 Nov-Dec, 7(6), 481 - 94 Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2 . Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor; Ozturk SS et al.; The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3) . The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA) . Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied . Cell growth was enhanced and cell death was decreased by increasing the serum level . The growth rates followed a Monod-type model with serum being the limiting component . Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations . Amino acid metabolism was slightly influenced by the serum level . Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation . Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation . Cell growth rate was optimal at pH 7.2 . Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2 . Metabolic rates for glutamine and ammonia were also higher below pH 7.2 . The consumption or production rates of amino acids followed the glutamine consumption very closely . Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH . Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH . The oxidative phosphorylation accounted for about 60% of total energy production . This contribution, however, increased at low pH values to 76% . The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20 . A 2-fold increase in specific antibody production rates was observed at pH values below 7.2 . Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained. Biotechnol Prog, 1991 Nov-Dec, 7(6), 471 - 80 Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1 . Analysis of data from controlled batch reactors; Ozturk SS et al.; A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration . The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods . The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase . Intracellular protein and RNA content followed a similar trend . Cell growth stopped when the glutamine in the medium was depleted . Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion . Ammonia and lactate production followed closely glutamine and glucose consumption, respectively . Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed . The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation . The antibody was produced at a constant rate in both the exponential and decline phases of growth . The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards. J Biotechnol, 1991 Nov, 21(1-2), 173 - 85 Enzyme sensor-FIA-system for on-line monitoring of glucose, lactate and glutamine in animal cell cultures; Renneberg R et al.; Enzyme sensors for glucose, lactate and glutamine were connected via flow-injection analysis (FIA) devices to two different bioprocesses . They were used for on-line process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2 . The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems. Biotechnology (N Y), 1991 Nov, 9(11), 1100 - 2 Modular integrated fluidized bed bioreactor technology; Reiter M et al.; We describe the design and demonstrate the application of a modular integrated fluidized bed bioreactor system . Basically the system is a reactor vessel equipped with an extending cylinder and a liquid distributor plate . Instead of an external recirculation loop, as used in existing fluidized bed systems, a low shear stress impeller is used as the recirculation pump . The system has several unique features, such as modular exchangeable elements, efficient oxygenation and the option of operating as a stirred tank-, a packed bed- or a fluidized bed reactor . An example of a fluidized bed run using CHO-K1 cells is shown . Under standard culture conditions a 100-fold increase in cell density (up to 1.2 x 10(8) cells/ml) was achieved. Glycoconj J, 1991 Oct, 8(5), 414 - 23 Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry; Muthing J et al.; YAC-1 cells were propagated in bioreactors in 1 l and 7.5 l volumes . The cells were metabolically labelled with D-{1-14C}galactose and D-{1-14C}glucosamine . The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a . Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside . As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b . The structural heterogeneity of these gangliosides was caused by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups . Disialogangliosides were detected only in low amounts and will be the subject of further investigation . A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer . The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone . No cross-reaction with GM1b or GgOse4Cer was observed. Antonie Van Leeuwenhoek, 1991 Oct-Nov, 60(3-4), 133 - 43 Quantitation of microbial metabolism; Sonnleitner B; Quantitation is a characteristic property of natural sciences and technologies and is the background for all kinetic and dynamic studies of microbial life . This presentation concentrates therefore on materials and methods as tools necessary to accomplish a sound, quantitative and mechanistic understanding of metabolism . Mathematical models are the software, bioreactors, actuators and analytical equipment are the hardware used . Experiments must be designed and performed in accordance with the relaxation times of the biosystem investigated; some of the respective consequences are discussed and commented in detail . Special emphasis is given to the required density, accuracy and reproducibility of data as well as their validation. Cytotechnology, 1991 Oct, 7(2), 63 - 74 The medium cycle bioreactor (MCB): monoclonal antibody production in a new economic production system; Kempken R et al.; The perfusion mode of a continuous cell culture bioreactor was modified to establish a closed loop system . Eighty percent of the spent medium was re-used twice . The medium cycle bioreactor unit was operated sterile and uncomplicated without a technical retention system for the high molecular weight substances . Therefore, only 20% of the actual medium was necessary to run the recycling process . During seven days culture time in a two liter scale 5 grams of IgG1 type monoclonal antibody was produced . During that period the cell specific productivity was constant . Renewal of proteins was omitted because the protein content in the system persisted at a high level . Therefore, self-conditioning substances of the cells were retained in the system as well as the expensive medium components (proteins with catalytic or stimulating function) . Seventy to 80% of medium costs and medium quantity were saved for each medium recycling step . Only cheap metabolites that are consumed by the cells had to be supplemented . Uptake rates of glucose and amino acids were calculated to establish a suitable supplementation mixture for the recirculated medium. Enzyme Microb Technol, 1991 Oct, 13(10), 822 - 7 Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells; Kompier R et al.; Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier . When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached . Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier . After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium . At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached . In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained . These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells. J Biotechnol, 1991 Oct, 20(3), 249 - 61 Monitoring of the production of monoclonal antibodies by hybridomas . Part II: Characterization and purification of acid proteases present in cell culture supernatant; van Erp R et al.; An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium . The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5 . Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants . These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis. J Biotechnol, 1991 Oct, 20(3), 235 - 48 Monitoring of the production of monoclonal antibodies by hybridomas . Part I: Long-term cultivation in hollow fibre bioreactors using serum-free medium; van Erp R et al.; The long-term cultivation of hybridoma cells in hollow fibre bioreactors using serum-free medium, was monitored with respect to quantitative and qualitative aspects of the produced mAbs, cell viability, LDH and proteolytic activity . During the culture periods of hybridoma cells producing mAb OT-1C and 3A, the mAb concentration showed a decreasing trend with a concomitant increase of IgG fragments . The major IgG fragments did not bind the antigen and the molecular weights were significantly different from the corresponding IgG heavy and light chains . In addition, a good correlation was found between cell lysis, the presence of acid protease(s) and IgG fragments . The physicochemical and immunochemical properties of the "intact" mAbs (such as molecular weights, IEF patterns and affinity) did not change significantly during the culture period. Anal Chem, 1991 Sep 15, 63(18), 1906 - 9 Operation of ion-selective electrode detectors in the sub-Nernstian/linear response range: application to flow-injection/enzymatic determination of L-glutamine in bioreactor media; Matuszewski W et al.; A novel approach for eliminating positive errors from endogenous ionic interferences when using ion-selective electrodes as detectors in flow-injection enzyme-based blosensing configurations is described . The method involves using a high background level of interfering ions in the sample diluent/carrier stream to convert the normally logarithmic potentiometric sensor into a linear detector over a given concentration range of primary ions . A split-stream single-detector arrangement provides a convenient means to compensate for varying levels of background interferent ions in the injected samples . One portion of the split stream passes directly to the ion-electrode detector, yielding a signal linearly related to the concentration of endogenous primary ions in the sample . The second portion of the split sample is delayed while passing through an immobilized enzyme that generates electrode detectable primary ions in proportion to the concentration of the substrate analyte in the sample . Two linear equations with two unknowns describe the twin potentiometric responses observed . The concept is demonstrated by the accurate determination of L-glutamine in hybridoma bioreactor media via the use of an ammonium-ion-selective membrane electrode detector and immobilized glutaminase enzyme. Cytotechnology, 1991 Sep, 7(1), 33 - 8 Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: effect of nutrient concentration; Franek F et al.; The iron-rich (500 microM ferric citrate) protein-free supplement was added to six different basal media . Cell growth and monoclonal antibody production of a mouse-mouse hybridoma were investigated in 1.3 1 batch cultures performed in a laboratory bioreactor with automatic control of pH and dissolved oxygen concentration . RPMI 1640 served as the control medium . Fortification of the basal medium by balanced mixtures of amino acids and vitamins showed higher positive effect than daily supplementation by glucose and glutamine . Strongly fortified medium, based on RPMI 1640, was found superior to other basal media . The viability index increased by a factor of 3.04 and the total antibody production by a factor of 2.82, relative to the control. Biotechnol Appl Biochem, 1991 Aug, 14(1), 60 - 8 Human red blood cells as bioreactors for the inactivation of harmful xenobiotics; Fazi A et al.; Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione . This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione . By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state . These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells . These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles. Virology, 1991 Aug, 183(2), 739 - 46 Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties; Kool M et al.; Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors . This configuration mimicked the serial undiluted passaging of virus, which is known to result in plaque-morphology mutants . Restriction enzyme analysis of DIP-containing preparations of extracellular virus showed the presence of many DNA fragments in less than equimolar amounts . These fragments were colinear on the physical map of AcNPV and extended from map position 1.7 to 45 . These DIPs thus lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes . The existence of DIPs was confirmed by electron microscopy, where virions were observed with reduced length . Among the less than equimolar fragments in DIP-containing preparations, fragments were observed linking sequences from map positions 1.7 and 45 via a TGTT linker of unknown origin . The DIPs could not be plaque-purified and needed standard (helper) virus to replicate; DIP-containing preparations interfered with standard virus replication in an interference assay, which explained the reduction in productivity of an AcNPV expression vector-insect cell system in continuous bioreactor operations . The origin of these DIPs and their possible generation mechanism are discussed. Trends Biotechnol, 1991 Aug, 9(8), 279 - 84 Strategies for improving plasmid stability in genetically modified bacteria in bioreactors; Kumar PK et al.; Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability . A wide range of strategies have been reported for improving the stability of recombinant organisms . A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors. ASAIO Trans, 1991 Jul-Sep, 37(3), M337 - 8 A hybrid artificial liver system . Function of cultured monolayer pig hepatocytes in plasma from hepatic failure patients; Uchino J et al.; To produce a new hybrid artificial liver system, cultured pig hepatocytes were used in this study . Hepatocytes from normal healthy pigs were cultured in multiwell dishes . From the third day of cultivation, hepatocytes were divided into three groups according to the media used (Group 1, L-15; Group 2, normal human plasma; Group 3, plasma from hepatic failure {HF} patients) . Measurements of cellular function of the cultured pig hepatocytes included metabolic activity (gluconeogenesis and ureogenesis), DNA content, and amino acid changes in the plasma . The ability to provide gluconeogenesis and ureogenesis by the cultured hepatocytes in HF plasma was maintained for 3 days, equal to that observed in Groups 1 and 2 . DNA content was no different in the three groups . Elevated amino acid levels in the HF plasma, Phe, Met, Lys, and Gly, were significantly reduced by the cultured hepatocytes . The results indicate that the use of primary cultured pig hepatocytes is a step toward a hybrid artificial liver system, and a promising candidate as a bioreactor. Appl Microbiol Biotechnol, 1991 Jul, 35(4), 440 - 6 Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs; Haumont M et al.; The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated . Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites . The performance of the bioreactor was optimized with the drug 3'-azido-3'-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase . Calcium (12 mM) could optimally improve the stability of microsomes entrapped in alginate beads . Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity . The determination of apparent Km and Vmax revealed that AZT was a better substrate for the immobilized enzyme than free microsomes . The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes . This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest. J Biotechnol, 1991 Jul, 19(2-3), 241 - 57 Polyvinyl alcohol and polyethylene glycol as protectants against fluid-mechanical injury of freely-suspended animal cells (CRL 8018); Michaels JD et al.; Two identical bioreactors run in parallel were used to examine the phenomenological characteristics of two additives, polyethylene glycol (PEG) and polyvinyl alcohol (PVA), used as protectants against fluid-mechanical cell damage . Cell-protecting ability was evaluated by comparing apparent cell growth rates of freely suspended CRL-8018 hybridoma cells cultured in serum-free medium under surface aerated conditions whereby cell damage is due to bubble entrainment and breakup . PEG of various molecular weights was used to determine whether the size of the polymer has significant effects on PEG's cell-protecting capabilities . All the PEG's with molecular weights larger than 1400 showed similar protective effects . The effect of PEG concentration was then evaluated and results showed that concentrations greater than 0.05% w/v did not significantly improve the cell-protecting properties . Direct comparisons made between the PVA, PEG, and pluronic F68 as cell protectants showed that PEG protected cells better than F68 and that PVA provided even better protection than PEG . The mechanism of protection, fluid-mechanical or biological in nature, was examined by growing the cells in additive from the beginning of the experiment (long-term exposure), or adding the additive after the cells had been agitated at rates detrimental to the cells (short-term exposure) . In agreement with results reported previously on PEG and F68, fast-acting protection was seen . This implies a fluid-mechanical rather than a biological protection mechanism . In an attempt to correlate interfacial properties of the resulting media with shear protection, interfacial tension and viscosity measurements of all the media were made . On the basis of these measurements, we find no definitive correlations for evaluating these additives' cell-protecting capabilities. Appl Environ Microbiol, 1991 Jun, 57(6), 1602 - 8 Performance characterization of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4; Folsom BR et al.; Pseudomonas cepacia G4 grown in chemostats with phenol demonstrated constant specific degradation rates for both phenol and trichloroethylene (TCE) over a range of dilution rates . Washout of cells from chemostats was evident at a dilution rate of 0.2 h-1 at 28 degrees C . Increased phenol concentrations in the nutrient feed led to increased biomass production with constant specific degradation rates for both phenol and TCE . The addition of lactate to the phenol feed led to increased biomass production but lowered specific phenol and TCE degradation rates . The maximum potential for TCE degradation was about 1.1 g per day per g of cell protein . Cell growth and degradation kinetic parameters were used in the design of a recirculating bioreactor for TCE degradation . In this reactor, the total amount of TCE degraded increased as either reaction time or biomass was increased . TCE degradation was observed up to 300 microM TCE with no significant decreases in rates . On the average, this reactor was able to degrade 0.7 g of TCE per day per g of cell protein . These results demonstrate the feasibility of TCE bioremediation through the use of bioreactors. Curr Opin Biotechnol, 1991 Jun, 2(3), 408 - 12 Integrated product formation and recovery; Daugulis AJ; As continues to be demonstrated, the in situ recovery of selected products froma bioreactor can have a significant positive impact on production . The strategies that are focused on here are: aqueous two-phase biocatalysis; non-aqueous biocatalysis; and membrane-enhanced biocatalysis . Additional fundamental understanding of molecular partitioning and biocatalytic activity in these environments will facilitate the rational selection of the components involved in these processing strategies. Curr Opin Biotechnol, 1991 Jun, 2(3), 375 - 9 Large-scale mammalian cell culture: methods, applications and products; Spier RE; Animal cell cultures are used to generate products of enormous biotechnological value . These systems rely on conventional manufacturing techniques using organisms that are the result of either cell fusions or genetic engineering . A wealth of new techniques has allowed improvements and developments to be made in culture medium composition, cell modification, and bioreactor design and operation . This progress is expected to be commercially exploited as new products reach the market place. Curr Opin Biotechnol, 1991 Jun, 2(3), 365 - 9 Large-scale insect cell culture: methods, applications and products; Goosen MF; The primary development in large-scale insect cell culture over the past year has been the continuing accumulation of documented evidence (fundamental and applied) that conventional aerated stirred-tank and air-lift bioreactors may be employed for insect cell cultivation and recombinant protein production, provided that air sparging, agitation, and the addition to the medium of Pluronic F-68 and methyl cellulose polymers are carefully controlled. J Biotechnol, 1991 Jun, 19(1), 99 - 110 Solasodine production from self-immobilised Solanum aviculare cells; Tsoulpha P et al.; Procedures were developed for 'self-immobilisation' of Solanum aviculare cells to eliminate the need for artificial immobilisation supports . Depending on the cytokinin used in liquid medium, compact aggregates 0.4-2.0 cm in diameter were formed without dispersed cells also being present . Histochemical analysis showed that the aggregates were structurally organised to facilitate nutrient transport . Growth, sugar uptake and solasodine production were measured in shake-flask cultures . Most of the product was stored in the aggregates to reach a maximum concentration of 0.3% dry weight; this is between 1.5 and 10 times the levels reported for suspended cells under similar conditions . A substantial amount of solasodine was produced after growth ceased . The maximum rate of solasodine production was about 0.22 mg g-1 d-1 . A simple air-driven bioreactor was tested for culture of the aggregates; solasodine yields were comparable to those measured in shake flasks. Anal Biochem, 1991 Apr, 194(1), 16 - 24 Flow injection analysis of lactose using covalently immobilized beta-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated Fractogel support; Narinesingh D et al.; Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors . These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support . The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase . The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid . Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996) . Between 30 and 40 milk samples/h can be analyzed . Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices . The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed. Cell Biophys, 1991 Apr, 18(2), 99 - 121 Possible role of cell cycle-dependent morphology, geometry, and mechanical properties in tumor cell metastasis; Needham D; Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream . Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle . In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle . Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells . As the cell cycle progressed at 37 degrees C, an increase in cell volume from 1400 microns 3 to 5700 microns 3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the "older" cells . Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells . It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line . For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle . Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system. Biotechnology (N Y), 1991 Apr, 9(4), 367 - 71 Bioconversion of n-octane to octanoic acid by a recombinant Escherichia coli cultured in a two-liquid phase bioreactor; Favre-Bulle O et al.; The alk genes from the catabolic OCT plasmid of Pseudomonas oleovorans, which encode the enzymes involved in the oxidation of n-alkanes to carboxylic acids, were introduced into E . coli W3110 . The resulting recombinant converts n-octane in a two-liquid phase medium into the corresponding alkanoate and excretes this compound into the aqueous phase . The rate of octanoic acid production by the recombinant E . coli is equal to or better than the alkane oxidation rate of P . oleovorans, suggesting that two-liquid phase fermentations with E . coli might have future industrial applications. Magn Reson Med, 1991 Mar, 18(1), 181 - 92 Iteration of hybridoma growth and productivity in hollow fiber bioreactors using 31P NMR; Gillies RJ et al.; Applications of nuclear magnetic resonance (NMR) spectroscopy to isolated or cultured mammalian cells have been limited because of technical difficulties in maintaining cultures at the extremely high densities required by NMR . Among the well-engineered systems available for such analyses, hollow fiber bioreactors (HFBRs) can maintain the greatest cell density . This attribute of HFBRs makes them ideal for application to NMR-based studies . These systems are currently being applied in biotechnology, where they are used for the production of mammalian cell-derived products, such as monoclonal antibodies . In this paper, the application of a HFBR system designed especially for NMR-based investigations is described . Performance of this system is monitored by NMR and the resulting stability and density of hybridoma cultures are reported . The resulting signal-to-noise per unit time is the highest seen to date for a mammalian cell system. Z Naturforsch {C}, 1991 Mar-Apr, 46(3-4), 204 - 9 Marine biosurfactants, II . Production and characterization of an anionic trehalose tetraester from the marine bacterium Arthrobacter sp . EK 1; Passeri A et al.; Within a screening for biosurfactants we could isolate various n-alkanes utilizing marine bacteria which were capable of synthesizing glycolipids . One strain was identified as Arthrobacter sp . EK 1 which produced trehalose lipids . After purification by column and thick layer chromatography the main fraction, an anionic 2,3,4,2'-trehalose tetraester, was obtained . The chain lengths of fatty acids ranged from 8 up to 14, furthermore succinate could be detected . Since the place of substitution of succinate has so far not been cited in literature, a definitive structural elucidation was carried out chemically by hydroboration and by 1H, 2D1H, 13C and 13C-1H correlation NMR measurements . All investigations confirmed the exact position of succinate at C 2 atom of trehalose . After improvement of growth conditions the production of the trehalose tetraester increased up to 4.8 milligrams during a fermentation in 20 l bioreactor under nitrogen limitation. Biotechnol Prog, 1991 Mar-Apr, 7(2), 130 - 9 Transport and kinetics in sandwiched membrane bioreactors; Jeong YS et al.; A bioreactor in which living yeast cells are sandwiched between an ultrafiltration membrane and a reverse osmosis membrane was constructed, and experiments were performed for the conversion of substrate glucose to product ethanol . A set of equations that include both transport through a series of barrier layers and bioreaction rate were developed to predict the performance of the sandwich bioreactor . The above equations were solved by using numerical values for the transport parameter and the bioreaction rate constant, and the results are compared with the experimental data. Appl Microbiol Biotechnol, 1991 Mar, 34(6), 726 - 9 A hybrid bioreactor for high density cultivation of plant cell suspensions; Kim DI et al.; A hybrid bioreactor was developed for the production of secondary metabolites from high density cultivation of plant cell suspensions . Some of the advantages of both air-lift and cell-lift by agitation were combined . The addition of a decanting column also made it possible to run a perfusion system for high density culture or to run a two-stage culture efficiently . Cell growth and the production of berberine from Thalictrum rugosum in the hybrid bioreactor are reported in this paper . A cell density up to 31 g/l was obtained by perfusion without any problems in mixing or loss of cell viability and the specific berberine productivity was comparable to that in shake flasks . The maximum berberine concentration was 88 mg/l at 3 weeks of operation and declined thereafter. Biotechnol Prog, 1991 Mar-Apr, 7(2), 85 - 92 Propagation of recombinant vaccinia virus in HeLa cells: adsorption kinetics and replication in batch cultures; Chillakuru RA et al.; The influence of various culture parameters on infection and replication of recombinant vaccinia virus in HeLa cells was examined during various phases of viral replication . A modified form of the model of Valentine and Allison (Biochim . Biophys . Acta 1960, 40, 393-399) model was used to predict successfully the viral adsorption rates in cell suspensions . An experimentally determined aggregation factor, epsilon, was included in the model to account for deviations of the observed adsorption rates from those predicted by the earlier model . It was also shown that the ionic strength, ionic species, and serum proteins present in the medium significantly altered the adsorption kinetics of the virus . The lysosomotropic base chloroquine was found to enhance viral infection more than 2-fold during the penetration step of viral infection . It was also demonstrated that cells infected during the exponential growth phase gave higher viral yields than those infected during the lag or stationary growth phases and the initial viral MOI did not significantly alter viral yields . Finally, it was demonstrated that viral infection of HeLa cells grown in 4-L bioreactor batch cultures resulted in increased death and glucose uptake rates and significantly lower growth rates. Cytotechnology, 1991 Feb, 5(2), 129 - 39 Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor . Cultivation of human hepatoma Hep G2 in hollow fiber bioreactor; Liu JJ et al.; Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described . Hep G2 cells (a human hepatoma cell line) were cultivated in an Acusyst-P (Endotronic) with a total fiber surface area of 7.2 m2 6 x 1.2m2) to produce Hep G2 crude conditioned medium (CCM) . Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells . We have succeeded in growing the Hep G2 cells in an antibiotics- and serum-free IMDM medium, supplemented with 50 micrograms/ml of Hep G2 CCM protein at inoculation . The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS) . The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated . Hep G2 CCM (20-40 micrograms protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro . The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents. J Biotechnol, 1991 Feb, 17(2), 133 - 46 Pilot scale production of a Trichoderma reesei endo-beta-glucanase by brewer's yeast; Zurbriggen BD et al.; Endo-beta-glucanase I (EGI) of Trichoderma reesei was produced in laboratory and pilot scale using recombinant strains of "bottom-fermenting" Saccharomyces cerevisiae . The gene eg/1 was integrated in the chromosome or an expression cassette was inserted on a multicopy plasmid . Expression levels were compared in a laboratory scale bioreactor . The best EGI-producing strain was cultivated in pilot scale . Adsorbent treatment was used to remove endogenous yeast proteins and other impurities from the culture filtrate during concentration . Effective pilot scale one-step purification of the EGI protein was obtained using DEAE-Sepharose, on which EGI was weakly bound . The purified enzyme reacted with antibodies prepared against T . reesei EGI and catalyzed the hydrolysis of both insoluble and soluble substrates. J Immunol, 1991 Jan 1, 146(1), 250 - 6 Recombinant soluble human complement receptor type 1 inhibits inflammation in the reversed passive arthus reaction in rats; Yeh CG et al.; The human CR1 was genetically engineered by site directed mutagenesis into a truncated form which was secreted from transfected Chinese hamster ovary cells . This soluble recombinant CR1 (sCR1) was purified from the supernatants of the Chinese hamster ovary cells cultured in a hollow fiber bioreactor . sCR1 inhibits the C3 and C5 convertases of the classical and the alternative pathways in vitro . The ability of sCR1 to inhibit the immune complex-mediated inflammation in vivo was tested in a rat reversed passive Arthus reaction model . Administration of sCR1 at the dermal sites reduced the Arthus vasculitis in a dose-dependent manner as judged by both gross and microscopic examination, as well as by immunohistologic localization of C3 and C5b-9 neoantigen deposits . These data suggest that sCR1 inhibits the Arthus reaction by interrupting the activation of the C cascade, hence limiting the detrimental immune complex-induced tissue damage in vivo. Biotechnology, 1991, 17, 305 - 26 Optimization of the microenvironment for mammalian cell culture in flexible collagen microspheres in a fluidized-bed bioreactor; Vournakis JN et al.; Flexible, three-dimensional, collagen Microspheres have been developed to actively promote a natural, optimal microenvironment for large-scale tissue culture of mammalian cells . The transport of nutrients into and cell products out of the Microspheres is enhanced by forced convective flow, which is the result of the tumbling of Microspheres and the dynamic properties of media flow in the fluidized-bed bioreactor . The collagen Microspheres have important characteristics of composition and morphology essential for optimal cell-matrix and cell-cell interactions . These interactions lead to high cell density and productivity through the dynamic modification of the microenvironment by cell-derived extracellular constituents . The collagen and Microsphere/fluidized-bed system provides the means to control and optimize the diffusive and contact components of the cells' microenvironment . Adaptation of cells to this microenvironment often results in dramatic increases in cell-specific productivity . Production of biotherapeutics in this process can be routinely performed in serum-free media, often leading to high productivity and product quality. Biotechnology, 1991, 17, 107 - 18 Nuclear magnetic resonance spectroscopy of dense cell populations for metabolic studies and bioreactor engineering: a synergistic partnership; Dale BE et al.; Commercial exploitation of the fruits of recombinant DNA and cell fusion technologies is significantly limited by the lack of fundamental metabolic information on the cell lines of interest, whether these are plant, animal, insect, or microbial cells . NMR can help to provide this information and thereby improve bioreactor design and operation . However, in the case of on-line NMR of dense cell culture devices for metabolic studies, these devices are inherently heterogeneous bioreactors . To ensure that the metabolic information generated is reliable, a number of precautions should be taken . These are the same precautions that should be taken to ensure that commercial bioreactors operate in a reaction-controlled regime . Therefore, reactor engineering methodologies, particularly diffusion and reaction analyses and reaction monitoring by whole-cell NMR must go hand in hand, each extending, complementing, and validating the other. Appl Biochem Biotechnol, 1991 Spring, 28-29, 457 - 69 Performance of trickle-bed bioreactors for converting synthesis gas to methane; Kimmel DE et al.; Carbon monoxide, H2, and CO2 in synthesis gas can be converted to CH4 by employing a triculture of Rhodospirillum rubrum, Methanosarcina barkeri, and Methanobacterium formicicum . Trickle-bed reactors have been found to be effective for this conversion because of their high mass-transfer coefficients . This paper compares results obtained for the conversion of synthesis gas to CH4 in 5-cm- and 16.5-cm-diameter trickle-bed reactors . Mass-transfer and scale-up parameters are defined, and light requirements for R . rubrum are considered in bioreactor design. Folia Microbiol (Praha), 1991, 36(1), 75 - 80 Immobilization of Aspergillus clavatus in a membrane bioreactor; Belushkina IA et al.; The possibility of the immobilization of A . clavatus in a membrane bioreactor which contains the nuclear membrane is investigated . The immobilization of cells in this bioreactor permits to increase the time of the productive functioning of the cells and avoids the procedure of a biomass separation from the cultural liquid. Biotechnol Genet Eng Rev, 1991, 9, 327 - 67 Heterologous protein production by filamentous fungi; Jeenes DJ et al.; There are clearly many facets to successful production of heterologous proteins from filamentous fungi . The objectives are to exploit the natural ability of some species to secrete high levels of protein . The heterologous target proteins produced in a fungal host must be acceptable to the public and be economic to produce, i.e . the targets must be authentic (in structure and activity) and be produced in high yield to necessary levels of purity . The appearance of heterologous products from fungi on the market is testament to some success but, equally, there are considerable limitations in our ability to produce desired yields of many target proteins . We endorse the view of van den Hondel, Punt and van Gorcom (1991) that for the commercial production of heterologous proteins from filamentous fungi more information is required on transcriptional control, introns, mRNA stability and processing, translational efficiency, protein secretion, glycosylation and proteolysis . In addition, there is scope for yield improvement based on a better understanding of the physiology of growth/product secretion coupled to appropriate bioreactor operation . The authenticity of product is an aspect which will assume increasing importance, particularly for therapeutic proteins . The level at which the structures and functional activity of heterologous proteins are assessed will ultimately be determined by legislation . The analytical methods currently available are not always sufficient, for example, to reveal folded structures, and most proteins are not amenable to analysis by two-dimensional NMR . The authenticity of target heterologous proteins will also need to be assessed in relation to the glycosylation level and pattern . This is not easily done and explains the paucity of detailed information published to date on glycosylation of fungal proteins . Novel engineered proteins are already being produced from filamentous fungi where expression is an aid to investigation of structure-function relationships . Commercial production of such engineered proteins will require approval subject to a range of stringently applied tests and analyses . This imposes an even greater need to be able to specify and control, in a rational manner, the structures of recombinant proteins . The research needs for realization of improved yields are equally important in assuring authenticity of product . It is encouraging that progress is being made on all fronts, primarily with Aspergillus spp . and T . reesei, but also with other species, such as N . crassa. Adv Biochem Eng Biotechnol, 1991, 44, 65 - 95 Shear effects on suspended cells; Merchuk JC; Shear has been mainly considered in the technical literature as a destructive element, when applied to microorganisms and cells . Indeed, most of the research work addressing the subject aims at the identification of damaging levels of shear on a given culture . The present work is focused on the effects of shear on suspended cultures before the damaging levels are attained . Inspection of the literature reveals that shear may influence growth rate, cellular volume, metabolite production rate and distribution, and membrane permeabilities . Available devices for study and evaluation of shear effects on suspended cultures are described and critically reviewed . The review reveals the possibility of an influence of the liquid dynamics on the kinetics of the biochemical process . This is relevant for bioreactor design and scale up, and stresses the importance of using structural bioreactor models in order to describe the hydrodynamics of the system. Adv Biochem Eng Biotechnol, 1991, 44, 27 - 64 Membrane bioreactors: present and prospects; Chang HN et al.; Membrane bioreactors have a very handy in-situ separation capability lacking in other types of bioreactors . Combining various functions of membrane separations and biocatalyst characteristics of enzymes, microbial cells, organelles, animal and plant tissues can generate quite a number of membrane bioreactor systems . The cell retaining property of membranes and selective removal of inhibitory byproducts makes high cell density culture possible and utilizes enzyme catalytic activity better, which leads to high productivity of bioreactors . Enzyme reactions utilizing cofactors and hydrolysis of macromolecules are advantageous in membrane bioreactors . Anaerobic cell culture may be efficiently carried out in membrane cell recycle systems, while aerobic cultures work well in dual hollow fiber reactors . Animal and plant cells have much a better chance of success in membrane reactors because of the protective environment of the reactor and the small oxygen uptake rate of these cells . Industrial use of these reactors are still in its infancy and limited to enzyme and animal tissue culture, but applications will expand as existing problems are resolved. J Chem Technol Biotechnol, 1991, 51(3), 323 - 34 Heat transfer in bubble column and airlift bioreactors: Newtonian and non-Newtonian fermentation broths; Kawase Y et al.; A theoretical model has been developed for heat transfer in bubble column and airlift bioreactors, which is applicable for Newtonian and non-Newtonian fermentation media . The proposed model is based on a similarity betwe