Bull Exp Biol Med, 2000 Jun, 129(6), 595 - 7 Functional activity of hepatocytes in tissue fragments in a new bioreactor biological artificial liver; Solov'ev VV et al.; Functional activity of hepatocytes in a new bioreactor designed for culturing of liver tissue fragments under perfusion conditions was tested . Specific hepatic functions such as ammonium detoxification, urea and protein synthesis, and P-450-dependent metabolism of p-nitroanisole were maintained for 1.5 days . The bioreactor can be used as a bioartificial liver support apparatus. FEMS Microbiol Lett, 2001 Feb 20, 195(2), 127 - 32 Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli; Lee J et al.; Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 {pUDF-A81} . In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus . In a fed-batch bioreactor containing recombinant E . coli at high cell concentrations (OD(600)>200), the extracellular LFTase accumulated to 46000 U ml(-1) (approximately 2.0 g l(-1)) which was almost 40% of total (intra- and extracellular) recombinant LFTase . The synthesized recombinant enzyme was secreted soon after gene expression was induced by IPTG . Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed-batch cultures . When lactose was added by continuous feed mode, the secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level . Therefore the induced cell culture of recombinant E . coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis . In the case of the controlled feed of lactose, the maximum activities (U ml(-1)) of total and extracellular LFTase were nearly 100% and 70% higher, respectively. ALTEX, 1992, 9(1), 15 - 24 {Monoclonal antibodies: Comparative methods for in vitro production}; Fischer RW et al.; The growth of antibody producing hybridoma in the abdominal cavity of the mouse with subsequent harvesting of the ascites fluid is a simple method for the production of monoclonal antibodies . However, due to the massive painful stress of the animal, this method is no longer tolerated by the legislator . In search of alternative methods the scientist has the choice between different techniques: Static tissue culture, spin- or roller-cultures as well as the production in bioreactors (automated cell culture devices) . The presented work investigates the different methods and describes their applicability in a research lab . The results clearly show that for any desired amount of antibodies a production technique is available which allows the omission of the use of the "ascites-mouse". ALTEX, 1994, 11(4), 207 - 215 {Bioreactor for long-term maintenance of differentiated hepatic cell functions}; Gerlach J et al.; A culture model and a bioreactor construction for hepatocyte in vitro culture is described . The reactor is based on capillaries for hepatocyte immobilisation . Four discrete capillary membrane systems, each serving different purposes, are woven to create a three-dimensional framework for decentralized cell perfusion with low metabolite gradients and decentralized oxygenation and CO2 removal . The biochemical performance of reactors initially seeded with 2.5 x 109 pig hepatocytes was evaluated (pig albumin synthesis, midazolam- and lidocaine/MEGX metabolism, galactose elimination, enzymeleakage) . The specific construction of the reactor enables a use in pharmacology alternatively to animal experiments. ALTEX, 1994, 11(2), 85 - 91 {Production of monoclonal antibodies in chicken eggs}; Hlinak A et al.; The possibility to produce monoclonal antibodies in chicken eggs was shown . Knowledge of biochemical and biophysical parameters of eggs were the basic of the experiments . The cell clones produced 0.1 mg/ml of antibodies in the egg fluid . This method can be a alternative to the monoclonal antibody production in mouse ascites or in bioreactors. Cells Tissues Organs, 2001, 168(3), 126 - 36 Hepatocytes entrapped in alginate gel beads and cultured in bioreactor: rapid repolarization and reconstitution of adhesion areas; Falasca L et al.; The maintenance of the differentiated hepatocyte phenotype and its specific physiological properties is known to depend on several factors, such as chemical signals, cell-cell and extracellular matrix molecular interactions, as well as the use of three-dimensional matrices . The entrapment of hepatocytes within Ca-alginate at high cell density and the culture under continuous flow favour the development of three-dimensional organization and promote expression of the differentiated hepatic phenotype . This system could represent an improvement in hepatocyte cultivation for basic studies of liver physiology and metabolism; it could also be applicable in toxicology, hepatocyte transplantation or development of bioartificial organs . This report describes the effect of alginate entrapment and culture in a bioreactor on hepatocyte aggregate formation, with particular attention to the re-establishment of cell polarity, cell junctions and three-dimensional re-organization of the cytoskeleton . Oxygen supply and cell oxygen consumption rate were monitored in order to evaluate possible changes in hepatocyte energy requirement . Our data show that after only 6 h of perfusion in the bioreactor, actin and cytokeratin localize along the adhesion areas of the plasma membrane, in which reconstituted bile canaliculi were also observed . Moreover, the presence of connexin at the level of joined membranes of neighbouring cells suggests the establishment of gap junctions between hepatocytes . After the first 30 min of perfusion the oxygen consumption rate remained constant throughout the experimental period . Biochem, Eng . J. . 2001 Mar, 7(2), 157 - 162 Device for sterile online measurement of the oxygen transfer rate in shaking flasks; Anderlei T et al.; The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption . Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult . However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently . A newly developed measuring device eliminates this deficit . Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel. Biochem, Eng . J. . 2001 Mar, 7(2), 135 - 141 Out-of-phase operating conditions, a hitherto unknown phenomenon in shaking bioreactors; Buchs J et al.; One of the important parameters in characterising fermentations of aerobic microorganisms is the specific power consumption . A new method has been introduced which enables the accurate determination of the power consumption in shaking bioreactors . It is based on torque measurements in the drive and the appropriate compensation of the friction losses . Measurements of the power consumption revealed the phenomenon of the liquid being 'out-of-phase' for the first time for shaking bioreactors . This occurs at certain operating conditions and is characterised by an increasing amount of liquid not following the rotating movement of the shaker table, thus reducing the specific power consumption, mixing and the gas/liquid mass transfer . With respect to this, different hydrodynamic cases have to be distinguished . All these cases have in common, however, that the probability of 'out-of-phase' conditions increases with lower shaking diameters, lower filling volumes, larger number and sizes of baffles and higher viscosity . For unbaffled flasks with a nominal volume </=1l the 'out-of-phase' phenomenon is described in the form of a newly defined non-dimensional Phase number (Ph) . To avoid the (unidentified) development of a screening project in unfavourable directions or even its complete failure, researchers must be aware of the 'out-of-phase' phenomenon . The experimental protocols have to be carefully selected so that the occurrence of such unwanted hydrodynamic conditions is not possible under all experimental circumstances. Biochem, Eng . J. . 2001 Mar, 7(2), 121 - 125 Development of a shaking bioreactor system for animal cell cultures; Liu C et al.; The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors . It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory. Biochem, Eng . J. . 2001 Mar, 7(2), 117 - 119 Small-scale bioreactor system for process development and optimization; Girard P et al.; An agitated 12-well microtiter plate system with a working volume of 2ml was investigated for cell culture process development . Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability . The pH of the NaHCO(3)-buffered system can be adjusted by altering the carbon dioxide content of the gas phase . The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for rapid and precise pH measurements . For high throughputs, cell growth was assessed non-invasively using stable green fluorescent protein (GFP) expressing cells and a fluorescence plate reader . The setup is simple and inexpensive . The system can be automated and allows several hundred small-scale bioreactor experiments to be run in parallel. Biochem, Eng . J. . 2001 Mar, 7(2), 107 - 112 Mass transfer resistance of sterile plugs in shaking bioreactors; Mrotzek C et al.; One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug . The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel {Bioprocess Eng . 7 (1991) 123} . Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures . It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied . Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined . Instead of the oxygen transfer coefficient (k(O(2))), which is commonly found in the literature, the carbon dioxide diffusion coefficient (D(CO(2))) is used to describe the mass transfer resistance of the sterile plug . The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density . The gas exchange of aluminium-caps was not reproducible. Biochem, Eng . J. . 2001 Mar, 7(2), 99 - 106 Characterisation of the gas-liquid mass transfer in shaking bioreactors; Maier U et al.; The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium . The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask . This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters . The model agrees well with the experimental results . It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR). Biochem, Eng . J. . 2001 Mar, 7(2), 91 - 98 Introduction to advantages and problems of shaken cultures; Buchs J; Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades . In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view . The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors . Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner . Unfortunately, these kind of interactions are often overlooked or ignored by scientists . Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions . In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed. Biotechnol Prog, 2001 Jan-Feb, 17(1), 95 - 103 A method to evaluate the isothermal effectiveness factor for dynamic oxygen into mycelial pellets in submerged cultures; Silva EM et al.; Several models have been developed simulating O2 transfer in bioreactors, but three limitations are often found: (i) an inadequate kinetic representation of O2 consumption or wrong boundary conditions, (ii) unrealistic parameter values, and (iii) inadequate experimental systems . In our study we minimized those possible sources of error . Oxygen uptake rate, void fraction of the pellet, and external O2 mass transfer coefficient were experimentally obtained from bioreactor studies in which pellets of Gibberella fujikuroi were naturally formed . Michaelis-Menten kinetics and diffusion equations were used to describe the O2 consumption rate and to evaluate the effectiveness factor in dynamic mode . The nonlinear mathematical model proposed was solved by the orthogonal collocation technique . The O2 consumption rate in pellets of G . fujikuroi of 1.7-2.0 mm is only marginally inhibited by diffusion constraints under conditions tested . Simulation analysis showed that the effectiveness factor decreased as the Thiele modulus and pellet diameter increased . The proposed model was applied to experimental data reported for other fungal pellets and allowed to predict optimal conditions for O2 transfer into mycelial pellets. Biotechnol Prog, 2001 Jan-Feb, 17(1), 81 - 8 Dynamic optimization of fed-batch bioreactors using the ant algorithm; Jayaraman VK et al.; The ant colony algorithm, mimicking the cooperative search behavior of ants in real life, has been employed for the dynamic optimization of fed-batch bioreactors . To test the capability of this new heuristic algorithm, two well-known and extensively studied systems have been chosen . The algorithm rapidly converges to optimal feed rate profiles, which maximize the overall production of the desired product and the profits in a computationally efficient and robust manner . The optimal profiles evolved are easy to implement in plant operation . The algorithm compares favorably with the other known techniques. Enzyme Microb Technol, 2001 Feb 1, 28(2-3), 225 - 232 Enzyme immobilization on nylon-optimization and the steps used to prevent enzyme leakage from the support; Isgrove FH et al.; Because of its low cost, chemical and mechanical properties and ready availability in a number of different forms (e.g . powders, beads, nets, tubes, film, sheets, etc.) Nylon is an attractive matrix for enzyme immobilization . We report here a thorough evaluation of a protocol for enzyme immobilization on nylon film with relatively inexpensive and non-toxic reagents, involving acid hydrolysis, glutaraldehyde coupling and spacer molecules and employing beta-glucosidase and trypsin as model enzymes . We also describe steps for virtually eliminating enzyme leakage and non-specific binding . Individual steps in the procedure are simple and conditions flexible so, whilst evaluated in terms of binding proteins to nylon film, they should be applicable to other forms of nylon and suitable for binding most enzymes and proteins, including antibodies, providing a method having potential in both affinity chromatography/adsorption and in bioreactor applications. J Biotechnol, 2001 Feb 13, 85(2), 187 - 212 Bioreactor performance: a more scientific approach for practice; Lubbert A et al.; In practice, the performance of a biochemical conversion process, i.e . the bioreactor performance, is essentially determined by the benefit/cost ratio . The benefit is generally defined in terms of the amount of the desired product produced and its market price . Cost reduction is the major objective in biochemical engineering . There are two essential engineering approaches to minimizing the cost of creating a particular product in an existing plant . One is to find a control path or operational procedure that optimally uses the dynamics of the process and copes with the many constraints restricting production . The other is to remove or lower the constraints by constructive improvements of the equipment and/or the microorganisms . This paper focuses on the first approach, dealing with optimization of the operational procedure and the measures by which one can ensure that the process adheres to the predetermined path . In practice, feedforward control is the predominant control mode applied . However, as it is frequently inadequate for optimal performance, feedback control may also be employed . Relevant aspects of such performance optimization are discussed. J Biotechnol, 2001 Feb 13, 85(2), 175 - 85 Physiological responses to mixing in large scale bioreactors; Enfors SO et al.; Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor . Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones . It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation . The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes . The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor . Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone . It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor . Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor. Curr Opin Biotechnol, 2000 Apr, 11(2), 205 - 8 Production of viral vectors for gene therapy applications; Wu N et al.; Advances in cell culture engineering, cell metabolism, bioreactor design and operation, and downstream processing will all positively impact the bioprocessing of viral vectors . Design of appropriate vectors and tailoring of packaging cells to support more productive infections will be of paramount importance for production of high-titer and high-quality vectors . Furthermore, quantitative analysis of the infection parameters during virus propagation, such as time of infection, multiplicity of infection, the length of replication cycle, virus half-life, and burst size, will also be important to the process optimization . Finally, procedures for separation, purification and formulation of vector preparations have to be further developed. J Gene Med, 1999 Nov-Dec, 1(6), 433 - 40 Closed hollow-fiber bioreactor: a new approach to retroviral vector production; Pan D et al.; BACKGROUND: The ability to obtain high-titer and large quantities of retroviral vector production in a 'closed' system would have profound implications in clinical and experimental gene therapy . METHODS: We studied the cell growth and vector production of three retroviral packaging cell lines in a variety of conditions using hollow-fiber bioreactors designed as an 'artificial capillary system' (ACS) and enhanced with the application of a hermetically sealing device for sterile welding of connecting plastic tubings . Vector titer, fetal bovine serum (FBS) concentration, volume and the duration of productivity were assessed to optimize vector production . RESULTS: In this pilot study, we observed that retroviral vector production (frozen-and-thawed) from cultures containing as low as 2.5% FBS yielded titers up to 2.2 x 10(7) cfu/ml, 14.4-fold higher than titers obtained from control dish cultures . Up to 3 liters of vector supernatant were generated during a 2-month large-scale production run . There was a potential to double this volume of higher-titer supernatant by increasing the frequency of harvest . It seemed that a lower metabolic rate (i.e . lactate production) in the packaging cell culture was associated with higher vector producing ability . CONCLUSIONS: These data demonstrated the feasibility of producing retroviral vector with enhanced titers and clinically useful quantities in a 'closed' ACS . Thus a new approach for large-scale retroviral vector production is developed. Appl Microbiol Biotechnol, 2000 Dec, 54(6), 832 - 7 Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents; Ullah MA et al.; In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated . Substrates included wood chips, cereal grain, wheat husk and wheat bran . Suitability for transformation of chlorophenols depended on laccase production by the fungus . The greatest amount of laccase (<25 Units g(-1) substrate) was produced on wheat husk and wheat bran over 30 days colonisation . Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75-80% of pentachlorophenol (50 ppm) within 24 h . Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation . Addition of 8-12% yeast extract to the pellets increased laccase production five-fold . Colonised pellets were added to chlorophenol solutions in 200-4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. J Immunol Methods, 2001 Jan 1, 247(1-2), 205 - 16 Butyrate increases production of human chimeric IgG in CHO-K1 cells whilst maintaining function and glycoform profile; Mimura Y et al.; The influence of sodium butyrate on the production and glycosylation of recombinant mouse/human chimeric antibody by transfected CHO-K1 cells was investigated . We selected cells expressing 'wild-type' antibody with a human IgG3 heavy chain and a mutant of this molecule in which Phe 243 is replaced by Ala . These proteins have previously been shown to exhibit very different glycoform profiles with the mutant IgG being comprised of glycoforms having a high galactose and sialic acid content . Cell culture with 0-5 mM butyrate was shown to effect a 2-4-fold increase in antibody production whilst the induction of apoptosis was observed in a dose-dependent manner . The optimal butyrate concentration was observed to be 2 mM . The glycoform profile of each antibody produced in the presence of butyrate was analyzed by HPAEC-PAD and shown to be unchanged, relative to that produced in the absence of butyrate . Biological activity was evaluated by the ability of the antibodies to trigger superoxide generation, through Fc gamma RI, and shown to be independent of production in the presence or absence of butyrate . A similar increase in production was observed for a high antibody-producing cell line when expanded in a hollow fibre bioreactor under low-serum conditions (1%) . These results demonstrated that butyrate is of value for increasing the productivity of CHO-K1 for recombinant IgG and does not compromise either glycosylation or biological activity. J Pediatr Surg, 2001 Jan, 36(1), 146 - 51 Fetal tissue engineering: diaphragmatic replacement; Fauza DO et al.; BACKGROUND/PURPOSE: Prosthetic repair of congenital diaphragmatic hernia has been associated with high complication rates . This study was aimed at applying fetal tissue engineering to diaphragmatic replacement . METHODS: Fetal lambs underwent harvest of skeletal muscle specimens . Once expanded in vitro, fetal myoblasts were suspended in a collagen hydrogel submitted to controlled radial tension . The construct was then placed in a bioreactor . After birth, all animals underwent creation of 2 diaphragmatic defects . One defect was repaired with the autologous-engineered construct placed in between 2 acellular supporting membranes and the other with an identical construct but without any cells . Each animal was its own control (graft, n = 10) . Animals were killed at different time-points postimplantation for histologic examination . Statistical analysis was by analysis of variance (ANOVA) . RESULTS: Fetal myoblasts expanded up to twice as fast as neonatal cells . Hydrogel-based radial tension enhanced construct architecture by eliciting cell organization within the scaffold . No eventration was present in 4 of 5 engineered constructs but in 0 of 5 acellular grafts (P<.05) . At harvest, engineered constructs were thick and histologically resembled normal skeletal muscle, whereas acellular grafts were thin, floppy, and showed low cell density with increased fibrosis . CONCLUSIONS: Unlike acellular grafts, engineered cellular diaphragmatic constructs are anatomically and histologically similar to normal muscle . Fetal tissue engineering may be a viable alternative for diaphragmatic replacement. Cell Transplant, 2000 Sep-Oct, 9(5), 711 - 5 Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of a bioartificial liver; Enosawa S et al.; Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals . To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity . After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes . The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 x 10(7) to 4 x 10(9) cells . Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient . Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 x 10(9) GS-HepG2 cells was almost equivalent to 5 x 10(8) porcine hepatocytes inoculated into the circulatory flow bioreactor . Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-alpha (TNF-alpha) . Cytokine removal therefore promises to be another useful property of bioreactor cells. FEMS Microbiol Ecol, 2001 Jan, 34(3), 213 - 220 Single strand conformation polymorphism monitoring of 16S rDNA Archaea during start-up of an anaerobic digester; Leclerc M et al.; A laboratory-scale continuously mixed anaerobic digester was inoculated with a mix of anaerobic sludge and fed with glucose . The start-up strategy was progressive and chemical analyses were done to evaluate digester performance from day 1 to day 107 . In parallel, Archaeal community dynamics were monitored by SSCP analysis of the V3 region of 16S rDNA genes and further characterized by partial sequencing of 16S rDNA genes . At day 1 the inoculum contained at least five distinct Archaeal peaks close to known methanogenic species . The dominant peak was very close to Methanosaeta concilli, the remaining species being members of the Methanobacteriales and Methanomicrobiales . A rapid shift of the Archaeal population was observed during the experiment . At day 21 Methanobacterium formicicum, which was not detected at day 1, became the dominant methanogenic species in the bioreactor and remained so until the end of the experiment. Biotechnol Bioeng, 2001 Feb 5, 72(3), 369 - 73 Effect of airflow rate on yields of Steinernema carpocapse Az 20 in liquid culture in an external-loop airlift bioreactor; Neves JM et al.; Maximization of the contact between males and females is a key factor in the production of the nematode Steinernema carpocapsae in a bioreactor.%The influence of the airflow rate in male and female distribution and mass production in an external-loop bioreactor with a deceleration zone was studied . When operating at an airflow rate of 0.05 vvm, a high retention of females in the deceleration zone of the bioreactor was observed and a larger nematode productivity was obtained . At this aeration rate there was a higher proportion of males in that zone, which together with the lower circulation rate, increases the probability of encounters, thereby explaining the increase in productivity . Biotechnol Bioeng, 2001 Feb 5, 72(3), 346 - 52 Low-cost microbioreactor for high-throughput bioprocessing; Kostov Y et al.; The design of a microbioreactor is described . An optical sensing system was used for continuous measurements of pH, dissolved oxygen, and optical density in a 2 mL working volume . The K(L)a of the microbioreactor was evaluated under different conditions . An Escherichia coli fermentation in both the microbioreactor and a standard 1 L bioreactor showed similar pH, dissolved oxygen, and optical density profiles.%The low cost of the microbioreactor, detection system, and the small volume of the fermentation broth provide a basis for development of a multiple-bioreactor system for high-throughput bioprocess optimization . Appl Microbiol Biotechnol, 2000 Nov, 54(5), 698 - 704 The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension; Singh S et al.; Shake-flask cultivation of T . lanuginosus strain SSBP on coarse corn cobs yielded beta-xylanase levels of 56,500 nkat/ml at 50 degrees C, whereas other hemicellulases (beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) were produced at levels less than 7 nkat/ml . Cultivation on D-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs . The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor . On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT . At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation . The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production . Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T . lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production . Rheological difficulties precluded cultivation on corn cobs in the bioreactor . Volumetric xylanase productivities of 1,060,000 nkat/l x h and 589,000 nkat/l x h obtained on beechwood xylan and xylose indicate that T . lanuginosus strain SSBP is a hyperxylanase producer with considerable industrial potential. Transgenic Res, 2000, 9(4-5), 279 - 99; discussion 277 Molecular farming of pharmaceutical proteins; Fischer R et al.; Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants . Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical proteins . Complex mammalian proteins can be produced in transformed plants or transformed plant suspension cells . Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressed proteins are functional and almost indistinguishable from their mammalian counterparts . The breadth of therapeutic proteins produced by plants range from interleukins to recombinant antibodies . Molecular farming in plants has the potential to provide virtually unlimited quantities of recombinant proteins for use as diagnostic and therapeutic tools in health care and the life sciences . Plants produce a large amount of biomass and protein production can be increased using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field . Transgenic plants can also produce organs rich in a recombinant protein for its long-term storage . This demonstrates the promise of using transgenic plants as bioreactors for the molecular farming of recombinant therapeutics, including vaccines, diagnostics, such as recombinant antibodies, plasma proteins, cytokines and growth factors. Hybridoma, 2000 Oct, 19(5), 407 - 12 Selection and isolation of cells for optimal growth in hollow fiber bioreactors; Gramer MJ et al.; Growth of a murine hybridoma in a hollow fiber microbioreactor was poor . This corresponded to slow initial growth in the Maximizer, a pilot scale hollow fiber bioreactor system . Medium screening experiments with the microbioreactor demonstrated that the slow growth was due to dialysis of low molecular weight serum components (under about 10 kDa) from the cell side of the fibers to the basal medium on the noncell side of the fibers . Better growth can be achieved by adding serum to both sides of the fibers, but this is an expensive option . As an alternative, the microbioreactor was used to select for a population of cells that did not require serum on both sides of the fiber for optimal growth . From this population, a stable subclone was isolated using limiting dilution followed by growth assessment in microbioreactors . The subclone was cultured in the Maximizer under conditions identical to the parental cell line . The subclone reached confluency in about 9 days compared with about 16 days for the parental cell line . At confluency, the subclone produced antibody at twice the rate of the parental cell line . These results demonstrate that the microbioreactor is a useful tool for quickly isolating subclones that are better suited for growth in a hollow fiber bioreactor. Yeast, 2001 Jan 15, 18(1), 19 - 32 Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool; Nissen TL et al.; The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/NADP(+) and has a significant influence on product formation in microorganisms . The enzyme pyridine nucleotide transhydrogenase, which can catalyse transfer of reducing equivalents between the two nucleotide systems, occurs in several organisms, but not in yeasts . The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomyces cerevisiae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii . We therefore cloned sth, encoding this enzyme, and expressed it under the control of a S . cerevisiae promoter in a strain derived from the industrial model strain S . cerevisiae CBS8066 . Anaerobic batch cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+) . A specific transhydrogenase activity of 4.53 U/mg protein was measured in the extracts from the strain expressing the sth gene from A . vinelandii, while no transhydrogenase activity could be detected in control strains without the gene . Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate . Since NADPH is used to convert 2-oxoglutarate to glutamate while glycerol formation increases when excess NADH is formed, this suggested that transhydrogenase converted NADH and NADP(+) to NAD(+) and NADPH . This was further supported by measurements of the intracellular nucleotide concentrations . Thus, the (NADPH/NADP(+)):(NADH/NAD(+)) ratio was reduced from 35 to 17 by the transhydrogenase . The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate . Also the biomass and ethanol yields were significantly lowered by the transhydrogenase . Am J Physiol Heart Circ Physiol, 2001 Jan, 280(1), H168 - 78 Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies; Papadaki M et al.; The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors . After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-microm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm . Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium . Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43 . Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies. Toxicology, 2000 Nov 23, 154(1-3), 31 - 44 In vitro toxicology in hepatocyte bioreactors-extracellular acidification rate (EAR) in a target cell line indicates hepato-activated transformation of substrates; Koebe HG et al.; In this article we introduce an in vitro model for hepato-mediated toxicity testing consisting of a Hepatocyte-Bioreactor connected to a microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells . The EAR in this system represented the metabolic activity of a tested cell line under the influence of bioreactor supernatant . Cyclophosphamide (CYCL), a well-known hepato-activated cytostatic drug was used as a model substrate because of its widespread clinical use . The predrug CYCL needed CYP 450 dependent activation to its active cytotoxic metabolite 4-OH cyclophosphamide . Primary pig hepatocytes from slaughterhouse organs were cultured in a collagen sandwich configuration in specially designed flasks and after 3 days introduced into a 50 ml recirculating perfusion system including 30 microg/ml CYCL . In a parallel open circuit, this bioreactor was connected to three perfusion chambers of a microphysiometer system housing 1.5 x 10(5) ZR 751 cells (breast tumor cell line) . Bioreactor supernatant including CYCL was pumped at 150 microl/min into the microphysiometer . The recorded EARs under CYCL influence were correlated to controls, which were set to be 100% . After 1 and 7 h of bioreactor supernatant perfusion, including activated CYCL, the ZR 751 cell line showed an EAR of 98.99%+/-3.15 (mean+/-SD) and 81 . 32%+/-10.18 (P<0.05), respectively, as compared to controls (bioreactor supernatant from the identical set-up without CYCL) . The inactivated predrug CYCL showed no effect on the EAR: Perfusion of medium with 30 microg/ml CYCL alone, excluding the bioreactor activation, resulted in an EAR of 100 . 11%+/-4.74 (mean+/-SD) after 7 h . Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of the predrug by the bioreactor. Enzyme Microb Technol, 2000 Dec, 27(10), 778 - 783 Performance of a miniaturized bioreactor in space flight: microtechnology at the service of space biology; Walther I I et al.; We describe here the performance and the use of microtechnology in a miniaturized bioreactor developed for the continuous cultivation of yeast cells, Saccharomyces cerevisiae, in microgravity . This bioreactor has been used on two Shuttle missions, where its functionality was successfully demonstrated . In the future, bioreactors will become a key element for long-term experiments, and would also be applied in the cultivation of mammalian cells or tissues for medical applications. Biotechnol Bioeng, 2001 Jan 20, 72(2), 231 - 43 Model for on-line moisture-content control during solid-state fermentation; Nagel FJ et al.; In this study we describe a model that estimates the extracellular (nonfungal) and overall water contents of wheat grains during solid-state fermentation (SSF) with Aspergillus oryzae, using on-line measurements of oxygen, carbon dioxide, and water vapor in the gas phase . The model uses elemental balances to predict substrate dry matter losses from carbon dioxide measurements, and metabolic water production, water used in starch hydrolysis, and water incorporated in new biomass from oxygen measurements . Water losses caused by evaporation were calculated from water vapor measurements . Model parameters were determined using an experimental membrane-based model system, which mimicked the growth of A . oryzae on the wheat grains and permitted direct measurement of the fungal biomass dry weight and wet weight . The measured water content of the biomass depended heavily on the moisture content of the solid substrate and was significantly lower than the estimated values reported in the literature . The model accurately predicted the measured overall water content of fermenting solid substrate during fermentations performed in a 1.5-L scraped drum reactor and in a 35-L horizontal paddle mixer, and is therefore considered validated . The model can be used to calculate the water addition required to control the extracellular water content in a mixed solid-state bioreactor for cultivation of A . oryzae on wheat. Biotechnol Bioeng, 2001 Jan 20, 72(2), 219 - 30 Temperature control in a continuously mixed bioreactor for solid-state fermentation; Nagel FJ et al.; A continuously mixed, aseptic paddle mixer was used successfully for solid-state fermentation (SSF) with Aspergillus oryzae on whole wheat kernels . Continuous mixing improved temperature control and prevented inhomogeneities in the bed . Respiration rates found in this system were comparable to those in small, isothermal, unmixed beds, which showed that continuous mixing did not cause serious damage to the fungus or the wheat kernels . Continuous mixing improves heat transport to the bioreactor wall, which reduces the need for evaporative cooling and thus may help to prevent the desiccation problems that hamper large-scale SSF . However, scale-up calculations for the paddle mixer indicated that wall cooling becomes insufficient at the 2-m(3) scale for a rapidly growing fungus like Aspergillus oryzae . Consequently, evaporative cooling will remain important in large-scale mixed systems . Experiments showed that water addition will be necessary when evaporative cooling is applied in order to maintain a sufficiently high water activity of the solid substrate . Mixing is necessary to ensure homogeneous water addition in SSF . Automated process control might be achieved using the enthalpy balance . The enthalpy balance for the case of evaporative cooling in the paddle mixer was validated . This work shows that continuous mixing provides promising possibilities for simultaneous control of temperature and moisture content in solid-state fermentation on a large scale. Biotechnol Bioeng, 2001 Jan 20, 72(2), 166 - 76 Classification of stability behavior of bioreactors with wall attachment and substrate-inhibited kinetics; Ajbar A; The biodegradation of pollutants in continuous operation when the microbial population exhibits wall attachment is studied . The proposed model for wall attachment assumes two morphological forms of the microbial cell connected by metamorphosis reactions with first order exchange kinetics . An analysis of the stability of the bioreactor, carried out using elementary principles of the singularity theory and continuation techniques, allows for classification in the multidimensional parameter space of the various stability behaviors exhibited by the reactor model, for both substrate-inhibited and Monod kinetics . The analysis also shows the enhanced stability behavior of the bioreactor due to wall attachment. Biotechnol Bioeng, 2001 Jan 20, 72(2), 136 - 43 A continuous membrane bioreactor for ester synthesis in organic media: II . Modeling Of MBR continuous operation; Carvalho CM et al.; A model was developed to describe the conversion degree in a membrane bioreactor (MBR) for the synthesis of short-chain esters as a function of the flow rate . The transesterification reaction was catalyzed by a recombinant cutinase of Fusarium solani pisi microencapsulated in reversed micelles of AOT/isooctane . The differences of product concentration in permeate and retentate together with the deactivation profiles led to an enzyme distribution evaluation that describes the experimental values attained . The model considers the bioreactor design as well as its hydrodynamics and the enzyme kinetics . The approach included the analysis of the MBR operation as a CSTR, a PFR, and a series of continuous reactors . The comparative efficiency of these reactor types is discussed . The enzyme distribution was estimated for all the cases . The best description was obtained considering a series of two CSTRs . The modeling results led to a re-evaluation of cutinase operational stability . Deactivation rates correlated very well with the hydrodynamic aspects of biocatalyst location. Biotechnol Bioeng, 2001 Jan 20, 72(2), 127 - 35 A continuous membrane bioreactor for ester synthesis in organic media: I . Operational characterization and stability; Carvalho CM et al.; The feasibility of continuous ester synthesis in a membrane bioreactor (MBR) by a recombinant cutinase from Fusarium solani pisi was investigated, using the optimal conditions previously attained by medium engineering . The objective was to analyze the MBR behavior as a differential or an integral reactor . The main component of the reactor was an anisotropic ceramic membrane with 15,000 NMWCO . The operating variables included the influence of substrates ratio and flow rate on the conversion degree and on the productivity . The highest conversion degree was obtained using 1M of hexanol and 0.1M of butyl acetate as acyl donor . The use of these substrate concentrations led to a conversion degree of 79.3% and a specific productivity of 41 g hexyl acetate/(d x g cutinase), when the permeate flow rate was 0.025 mL/min . The increase of flow rate to 0.4 mL/min decreased the conversion to 35.6%, although the productivity was enhanced to 294 g product/day x g enzyme . The MBR characterization involved the calculations of mass balance, recirculation rate, conversion per pass, number of cycles, and hydraulic residence time . The operational stability was also evaluated in a longterm experiment over 900 hours and the enzyme half-life was estimated to be approximately 2 years. J Air Waste Manag Assoc, 2000 Nov, 50(11), 1947 - 56 Construction and economics of a pilot/full-scale biological trickling filter reactor for the removal of volatile organic compounds from polluted air; Deshusses MA et al.; The design and the construction of an actual 8.7-m3 pilot/full-scale biotrickling filter for waste air treatment is described and compared with a previous conceptual scale-up of a laboratory reactor . The reactor construction costs are detailed and show that about one-half of the total reactor costs ($97,000 out of $178,000) was for personnel and engineering time, whereas approximately 20% was for monitoring and control equipment . A detailed treatment cost analysis demonstrated that, for an empty bed contact time of 90 sec, the overall treatment costs (including capital charges) were as low as $8.7/1000 m3air in the case where a nonchlorinated volatile organic compound (VOC) was treated, and $14/1000 m3air for chlorinated compounds such as CH2Cl2 . Comparison of these costs with conventional air pollution control techniques demonstrates excellent perspectives for more field applications of biotrickling filters . As the specific costs of building and operating biotrickling filter reactors decrease with increasing size of the reactor, the cost benefit of biotrickling filtration is expected to increase for full technical-scale bioreactors. ALTEX, 2000, 17(1), 3 - 10 {Liver cell culture in bioreactors for in vitro drug studies as an alternative to animal testing}; Zeilinger K et al.; An important consideration for the utilisation of in vitro culture models for studies on drug metabolism as an alternative to animal testing is the maintenance of a defined degree of cell differentiation . Thus, in vitro conditions reflecting as near as possible the in vivo situation of the cells within the whole organ are required . A bioreactor was developed for the cultivation of liver cells which allows the reorganisation of hepatocytes and non-parenchymal cells of the liver in coculture to form three-dimensional, tissue-like structures including extracellular matrix components produced by the cells . In this study, the vitality and metabolic activity of isolated rat hepatocytes was investigated over a two week culture period in bioreactors . The results show that after a reorganisation phase, the cells preserve specific functions, such as protein and urea synthesis capacity and specific cytochrome P450 activities during the culture period, with maximal values during the first week . Possible applications of the model in pharmaceutical industry are studies on metabolite patterns, enzyme induction, drug-drug-interactions, first pass effects and long-term toxicity of drugs. Trends Biotechnol, 2000 Dec, 18(12), 501 - 5 Industrial wastewater bioreactors: sources of novel microorganisms for biotechnology; Bramucci MG et al.; Microorganisms exist in nature as members of complex, mixed communities . The microbial communities in industrial wastewater bioreactors can be used as model systems to study the evolution of new metabolic pathways in natural ecosystems . The evolution of microbial metabolic capability in these bioreactors is presumably analogous to phenomena that occur in natural ecosystems . The microorganisms in these bioreactors compete for different carbon sources and constantly have to evolve new metabolic capabilities for survival . Thus, industrial bioreactors should be a rich source of novel biocatalysts. Biotechnol Prog, 2000 Nov-Dec, 16(6), 1044 - 54 Pressure and flux profiles in bead-filled ultrafiltration/microfiltration hollow fiber membrane modules; Dai XP et al.; A general mathematical model for the prediction of pressure, flow rate, and flux profiles in an ultrafiltration/microfiltration hollow fiber membrane module whose shell side is filled with beads has been developed . The model was studied for a variety of operational modes in such modules, e.g., ultrafiltration/microfiltration, permeate flow rate control, Starling flow (encountered in hollow fiber bioreactors), and tube-side elution (encountered in filtration-cum-chromatography processes), etc., with or without a bead-filled extended section at the permeate outlet . An algorithm is provided to determine the model parameters from experimental data using the model equations . The solutions developed have been used to study the uniformity of transmembrane pressure profile along the module length using a quantity called the uniformity factor alpha . This factor shows that the model can be a useful tool for achieving the desired module performance in a number of quite different applications . The model predicts successfully the nature of the transmembrane pressure profile and the solvent flux profile in situations that are quite different, namely, conventional ultrafiltration and Starling flow . The approach used in this study can also be adopted to develop a model for description of other operational modes such as backflushing and shell-side elution used in the processes of filtration-cum-chromatography . Those applications employing similar device configurations may also use this model to predict the pressure and flux profiles to facilitate the design of the process and the operation conditions. Biotechnol Prog, 2000 Nov-Dec, 16(6), 979 - 85 Fed-batch bioreactor strategies for microbial decolorization of azo dye using a Pseudomonas luteola strain; Chang JS et al.; A Pseudomonas luteola strain possessing azoreductase activity was utilized to decolorize a reactive azo dye (C . I . Reactive Red 22) with fed-batch processes consisting of an aerobic cell growth stage and an anaerobic fed-batch decolorization stage . The fed-batch decolorization was conducted with different agitation and aeration rates, initial culture volumes, dye loading strategies, and yeast extract to dye (Y/D) ratios, and the effect of those operation parameters on azo dye decolorization was evaluated . Dissolved oxygen strongly inhibited the azo reduction activity; thus aeration should be avoided during decolorization but slight agitation (around 50 rpm) was needed . With the periodical feeding strategy, the specific decolorization rate (v(dye)) and overall decolorization efficiency (eta(dye)) tended to increase with increasing feeding concentrations of dye, whereas substrate inhibition seems to arise when the feeding concentration exceeded 600 mg dye/L . In the continuous feeding mode, higher initial culture volume resulted in better eta(dye) due to higher biomass loading, but lower v(dye) due to lower dye concentration in the bioreactor . With a volumetric flow rate (F) of 25 mL/h, both v(dye) and eta(dye) increased almost linearly with the increase in the loading rate of dye (F(dye)) over the range of 50-200 mg/h, while further increase in F(dye) (400 mg/h) gave rise to a decline in v(dye) and eta(dye) . As the F was doubled (50 mL/h), the v(dye) and eta(dye) increased with F(dye) only for F(dye) < 80 mg/h . The best v(dye) (113.7 mg dye g cell(-)(1) h(-)(1)) and eta(dye) (86.3 mg dye L(-)(1) h(-)(1)) were achieved at F(dye) = 200 mg/h and F = 25 mL/h . The yield coefficient representing the relation between dye decolorized and yeast extract consumed was estimated as 0.8 g/g . With F(dye) = 75 mg/h, the Y/D ratio should be higher than 0.5 to ensure sufficient supply of yeast extract for stable fed-batch operations . However, performance of the fed-batch decolorization process was not appreciably improved by raising the Y/D ratio from 0.5 to 1.875 but was more sensitive to the changes in the dye loading rate. Biotechnol Prog, 2000 Nov-Dec, 16(6), 966 - 72 Two-liquid-phase slurry bioreactors to enhance the degradation of high-molecular-weight polycyclic aromatic hydrocarbons in soil; Villemur R et al.; High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments . The addition of a water-immiscible, nonbiodegradable, and biocompatible liquid, silicone oil, to a soil slurry was studied to promote the desorption of PAHs from soil and to increase their bioavailability . First, the transfer into silicone oil of phenanthrene, pyrene, chrysene, and benzo{a}pyrene added to a sterilized soil (sandy soil with 0.65% total volatile solids) was measured for 4 days in three two-liquid-phase (TLP) slurry systems each containing 30% (w/v) soil but different volumes of silicone oil (2.5%, 7.5%, and 15% {v/v}) . Except for chrysene, a high percentage of these PAHs was transferred from soil to silicone oil in the TLP slurry system containing 15% silicone oil . Rapid PAH transfer occurred during the first 8 h, probably resulting from the extraction of nonsolubilized and of poorly sorbed PAHs . This was followed by a period in which a slower but constant transfer occurred, suggesting extraction of more tightly bound PAHs . Second, a HMW PAH-degrading consortium was enriched in a TLP slurry system with a microbial population isolated from a creosote-contaminated soil . This consortium was then added to three other TLP slurry systems each containing 30% (w/v) sterilized soil that had been artificially contaminated with pyrene, chrysene, and benzo{a}pyrene, but different volumes of silicone oil (10%, 20%, and 30% {v/v}) . The resulting TLP slurry bioreactors were much more efficient than the control slurry bioreactor containing the same contaminated soil but no oil phase . In the TLP slurry bioreactor containing 30% silicone oil, the rate of pyrene degradation was 19 mg L(-)(1) day(-)(1) and no pyrene was detected after 4 days . The degradation rates of chrysene and benzo{a}pyrene in the 30% TLP slurry bioreactor were, respectively, 3.5 and 0.94 mg L(-)(1) day(-)(1) . Low degradation of pyrene and no significant degradation of chrysene and benzo{a}pyrene occurred in the slurry bioreactor . This is the first report in which a TLP system was combined with a slurry system to improve the biodegradation of PAHs in soil. Biochimie, 2000 Nov, 82(11), 1063 - 85 Reverse micelles as reaction media for lipases; Carvalho CM et al.; Reversed micelles are at the present time faced as common organic media to perform biocatalysis . They have been associated to the idea of a microreactor where the enzyme can be sheltered and protected from solvent detrimental effects . This simplistic idea led some investigators to ignore some basic understanding, such as the recognition of the enzyme-specific microenvironment and what the enzyme experiences inside the reversed micelle . To date the number of reactions catalyzed by lipases in reversed micelles is large . This review aims to highlight some of the fundamental aspects of the lipase microencapsulation as well as to resume the outstanding progress of the reversed micellar systems . The properties of the micellar microenvironment are reviewed and related to the lipases' performance both in terms of activity and stability . The heterogeneity of reversed micellar systems is discussed in relation to component distribution models and also to enzymatic kinetics . The new trends and the practical aspects where efforts should be centralized in order to spread out the micellar bioreactor technology over industrial processes are also discussed. Med Biol Eng Comput, 2000 Sep, 38(5), 583 - 90 Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system; Torgan CE et al.; The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts . These cells are responsive to a variety of environmental cues, including mechanical stimuli . The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e . chemical and electrical stimuli) are controlled . To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity . C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes . In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation . This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin . Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01) . Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes . In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation . These results may have implications for muscle growth and repair during spaceflight. Appl Microbiol Biotechnol, 2000 Oct, 54(4), 589 - 96 Biodegradation of atrazine in sand sediments and in a sand-filter; Goux SJ et al.; The potential of a microbial consortium for treating waters contaminated with atrazine was considered . In conventional liquid culture, atrazine and its two dealkylated by-products were equally metabolised by the microbial consortium . Transient production of hydroxyatrazine was observed during atrazine catabolism, indicating that the catabolic pathway was similar to the one reported for isolates capable of atrazine mineralisation . This consortium was then inoculated to sediments sampled from an artificial recharge site . These sediments were contaminated by atrazine and diuron and exhibited only a slow endogenous herbicide dissipation . Inoculated microorganisms led to extensive atrazine degradation and survived for more than 10 weeks in the sediments . A rudimentary bioreactor was then setup using a soil core originating from the same recharge site . Degrading microorganisms rapidly colonised the core and expressed their degrading activity . The efficiency of the bioreactor was improved in the presence of spiked environmental surface waters . Atrazine degraders thus possibly benefited from the other organic sources in developing and expressing their activity . The microbial consortium did not initially exhibit the capacity to degrade diuron, which was used as reference compound . No change in this characteristic was detected throughout the study. Adv Biochem Eng Biotechnol, 2000, 70, 1 - 33 The morphology of filamentous fungi; Kossen NW; The morphology of fungi has received attention from both pure and applied scientists . The subject is complicated, because many genes and physiological mechanisms are involved in the development of a particular morphological type: its morphogenesis . The contribution from pure physiologists is growing steadily as more and more details of the transport processes and the kinetics involved in the morphogenesis become known . A short survey of these results is presented . Various mathematical models have been developed for the morphogenesis as such, but also for the direct relation between morphology and productivity--as production takes place only in a specific morphological type . The physiological basis for a number of these models varies from thorough to rather questionable . In some models, assumptions have been made that are in conflict with existing physiological know-how . Whether or not this is a problem depends on the purpose of the model and on its use for extrapolation . Parameter evaluation is another aspect that comes into play here . The genetics behind morphogenesis is not yet very well developed, but needs to be given full attention because present models and practices are based almost entirely on the influence of environmental factors on morphology . This makes morphogenesis rather difficult to control, because environmental factors vary considerably during production as well as on scale . Genetically controlled morphogenesis might solve this problem . Apart from a direct relation between morphology and productivity, there is an indirect relation between them, via the influence of morphology on transport phenomena in the bioreactor . The best way to study this relation is with viscosity as a separate contributing factor. J Biotechnol, 2001 Nov 30, 84(2), 107 - 18 Characterization of bioreaction processes: aerobic Escherichia coli cultures; Guardia Alba MJ et al.; The simulation of a microbial transformation course is an important tool for the optimal design or characterization of industrial processes . Usually, models are developed to describe a specific part of the culture such as microbial growth and most of the time ignore the influence of physical and chemical environment on growth dynamics of the microorganism . In this work we propose a method which combines the description of the evolution of components involved in the bioprocess including biomass and the physical environment generated mainly by the bioreactor characteristics and operational conditions . Stoichiometric, kinetic, fluid dynamics and mass transfer models are linked to predict the course of the Escherichia coli culture under the influence of different experimental conditions and types of bioreactors . A set of 22 kinetic and physical parameters obtained from independent experiments and from literature are used in order to predict glucose, biomass, acetate, dissolved oxygen and CO(2) concentrations in airlift and stirred tank bioreactors. Hum Gene Ther, 2000 Nov 1, 11(16), 2297 - 300 Regulated cutaneous gene delivery: the skin as a bioreactor; Cao T et al.; Epidermal keratinocytes can secrete polypeptides into the bloodstream, and they can be easily expanded in culture and genetically modified . It is thus possible to use epidermal keratinocytes for the systemic delivery of transgene products . Here we review the development of epidermal secretory systems, from cultured keratinocytes to skin grafts and transgenic mouse models . We also discuss a gene-switch approach for regulated cutaneous gene delivery. Biochem, Eng . J. . 2000 Dec 1, 6(3), 201 - 205 Effects of sugar concentration on recombinant human alpha(1)-antitrypsin production by genetically engineered rice cell; Terashima M et al.; Productivity of recombinant human alpha(1)-antitrypsin (rAAT) with a genetically engineered rice cell using an inducible promoter has been studied by batch-wise and continuous production . A simple model explained the effect of proteases released from the disrupted cells on the rAAT degradation . Glucose concentration in the medium significantly affected the rAAT productivity in the continuous production, because the rAAT was induced by sugar depletion . When the fresh medium containing 5mM glucose was supplied to the continuous bioreactor, induction time was long and the productivity was low, indicating that the glucose concentration in the cells was high enough as to repress the promoter . When the glucose concentration in the fresh medium was reduced to 0.5mM, total amount of rAAT produced in 70h cultivation reached 6.7-7.6mg/g-dry cell, which was two times larger than the control medium without glucose. Biochem, Eng . J. . 2000 Dec 1, 6(3), 163 - 175 Extractive fermentation for improved production of endoglucanase by an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae using aqueous two-phase system; Sinha J et al.; Extractive aqueous two-phase fermentation of endoglucanase, a key enzyme for the conversion of cellulosic substances to fermentable sugars, from an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae is a meaningful approach for better production and simple recovery of this enzyme . A phase composition of 6.5% (w/w) dextran and 7.5% (w/w) polyethylene glycol 6000, having a partition coefficient of 2.89 and 1.31 for endoglucanase from an intergeneric fusant of T . reesei/S . cerevisiae and T . reesei (WT) (being a control in this study), respectively, was chosen for extractive fermentation of the enzyme . Endoglucanase production is higher in medium containing polyethylene glycol (PEG) 6000 than in medium without PEG 6000 . Comparative analysis of endoglucanase fermentation by fusant and T . reesei was carried out in shake culture and environment-controlled bioreactor conditions . The fusant produced 0.43U of endoglucanase (overall production: 0.34U) in the top phase of an aqueous two-phase system (ATPS), compared to 0.3U in medium without the phase system in shake culture . In a batch reactor, the endoglucanase level for the fusant in the top phase of ATPS was 0.49U (overall production: 0.40U), compared to 0.38U produced in medium without aqueous two-phase components . To corroborate this study, T . reesei produced 8.41U of endoglucanase (overall production: 5.96U) in the top phase of ATPS, compared to 7.18U in the medium without the phase system in shake culture . On the other hand, in a batch bioreactor, T . reesei produced 10.13U of endoglucanase (overall production: 6.90U) in the top phase of ATPS, compared to 8.56U of the enzyme in medium without aqueous two-phase components . The lower overall enzyme production by T . reesei in the two-phase system might be due to limitation in oxygen transfer to the dispersed phase where the enzyme is produced . A higher cell concentration and a reduced lag phase was obtained in ATPS, compared to a similar medium without phase forming polymers for both the intergeneric fusant of T . reesei/S . cerevisiae and T . reesei. Tissue Eng, 2000 Oct, 6(5), 519 - 30 Numerical model and experimental validation of microcarrier motion in a rotating bioreactor; Pollack SR et al.; The equations of motion for microcarriers in a rotating bioreactor have been formulated and trajectories obtained using numerical techniques . An imaging system was built to validate the results by direct observation of microcarrier trajectories in the rotating frame of reference . The microcarrier motion observed by this imaging system was in excellent agreement with the numerical predictions of that motion . In the rotating frame of reference, microcarriers with density greater than the surrounding fluid medium followed a circular motion relative to the culture medium combined with a persistent migration and eventual collision with the outer wall of the reactor . However, for microcarrier density less the fluid medium, their circular motion migrated toward the central region of the reactor . When multiple microcarrier beads that are lighter than water are inserted into the reactor, the centrally directed migration results in the formation of clusters that are stabilized by tissue bridges formed by osteoblasts seeded onto the microcarriers . This system offers unique opportunities to monitor tissue synthesis on microcarriers using real-time optical techniques and to optimize the bioreactor operating conditions for exploiting this technology to study early bone tissue synthesis in vitro. Tissue Eng, 2000 Oct, 6(5), 481 - 95 Hepatocyte encapsulation for enhanced cellular functions; Chia SM et al.; An efficient bioartificial liver-assisted device can sustain the lives of patients with acute liver failure . Among different configurations of the bioreactor design, hepatocyte encapsulation has important features that satisfy most requirements of the device . We have encapsulated rat hepatocytes in a two-layer polymeric membrane by complex coacervation using a simple setup and demonstrated enhanced cellular functions up to three times higher than those of the monolayer control . These microcapsules of the functioning hepatocytes have a 2- to 3-microm outer layer of synthetic polymer with 25% 2-hydroxyethyl methacrylate, 25% methacrylic acid, and 50% methyl methacrylate and an inner layer of positively charged modified collagen as a suitable substrate for the enhanced cellular functions . Permeable only to small molecules up to albumin, the microcapsules should allow unimpeded exchange of nutrients, oxygen, growth factors, and metabolites but prevent attack by immunoglobulins of the immune system, and no "skin effect" of the collagen has been observed . Mechanical properties of the microcapsules measured with a nano-indentation method suggest that the microcapsules should be suitable for use in a bioartificial liver-assisted device. Biomaterials, 2000 Dec, 21(24), 2575 - 87 Ex vivo synthesis of articular cartilage; LeBaron RG et al.; This review discusses modern methods used for the synthesis of articular cartilage ex vivo . The value of culturing articular chondrocytes as a monolayer and in three-dimensional lattices is discussed . Of particular interest are techniques involving seeding of chondrocytes onto synthetic, biodegradable, polymeric scaffolds, and natural materials, such as collagen and agarose . Also discussed is the use of bioreactors to modulate the fluid-flow-induced shear environment of cell-seeded scaffolds . Biodegradable scaffolds are central to the efforts to tissue engineer articular cartilage ex vivo . A review of salient efforts to design and use such scaffolds is presented, along with our thoughts on potential future improvements. J Environ Sci Health B, 2000 Nov, 35(6), 647 - 75 Fate of atrazine and chlorpyrifos during solid state fermentation--examination of processes; Judge DN et al.; Solid state fermentation (SSF) was investigated as a means to dispose of two commonly used pesticides, chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) . SSF experiments were carried out in bench-scale bioreactors (equipped with CO2 and volatile organic traps) containing a mixture of lignocellulosic materials and a radiolabeled pesticide . Ethyl acetate-extractable, alkali soluble, and alkali insoluble fractions were evaluated for radioactivity following a 60-d incubation period at 40 degrees C . The majority of the {2,6-pyridyl-14C}chlorpyrifos was associated with the ethyl acetate extract (about 74%), 17% was trapped as organic volatiles by polyurethane foam traps and < 0.5% of the chlorpyrifos was mineralized to CO2 . Only small amounts of the radioactivity were associated with alkali soluble (0.0003%) and alkali insoluble (0.3%) fractions . In the {14C-U-ring}atrazine bioreactors, very little of the radioactivity volatilized (<0.5%) and less than 0.5% was mineralized to CO2 . Approximately 57% of the applied radioactivity was associated with the ethyl acetate extract while 9% and 24% of the radioactivity was associated with the alkali soluble (humic and fulvic acids) and alkali insoluble fractions, respectively . Possible reaction mechanisms by which covalent bonds could be formed between atrazine (or metabolites) and humic substances were investigated . The issue of bound atrazine residue (alkali soluble fraction) was at least partially resolved . Oxidative coupling experiments revealed that formation of covalent bond linkages between amino substituent groups of atrazine residue and humic substances is highly unlikely. Biotechnol Bioeng, 2000 Dec 20, 70(6), 670 - 6 Effective production of a thermostable alpha-glucosidase from Sulfolobus solfataricus in Escherichia coli exploiting a microfiltration bioreactor; Schiraldi C et al.; A microfiltration (MF) membrane bioreactor was developed for an efficient production of a recombinant thermostable alpha-glucosidase (rSsGA) from Sulfolobus solfataricus MT-4 . The aim of the membrane bioreactor was to improve the control of the concentration of key components in the growth of genetic engineered microorganisms, such as Escherichia coli . The influence of medium composition was studied in relation to cell growth and alpha-glucosidase production . The addition of components such as yeast extract and tryptone resulted in a higher enzyme production . High cell density cultivation of E . coli BL21(DE3) on semidefined medium, exploiting a microfiltration bioreactor, was studied in order to optimize rSsGA production . In addition to medium composition, the inducer employed (either isopropyl beta-D-thiogalactopyranoside or lactose), the induction duration, and the cultivation mode influenced both the final biomass and the enzyme yield . The MF bioreactor allowed a cell concentration of 50 g/L dry weight and a corresponding alpha-glucosidase production of 11,500 U/L . The improvement obtained in the enzyme production combining genetic engineering and the microfiltration strategy was estimated to be 2,000-fold the wild-type strain. Biotechnol Bioeng, 2000 Dec 20, 70(6), 662 - 9 "Oxidative stress" response in submerged cultures of a recombinant Aspergillus niger (B1-D); Kreiner M et al.; A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, was investigated with regard to its susceptibility to "oxidative stress" in submerged culture in bioreactor systems . The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide or by high-dissolved oxygen tensions, was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD) . Batch cultures in the bioreactor were generally found to have maximum specific activities of CAT and SOD (Umg x protein(-1)) in the stationary/early-decline phase . Continuous addition of H2O2 (16 mmole L(-1) h(-1)), starting in the early exponential phase, induced CAT but did not increase SOD significantly . Gassing an early exponential-phase culture with O2 enriched (25 vol%) air resulted in increased activities of both SOD and CAT relative to control processes gassed continuously with air, while gassing the culture with 25 vol% O2 enriched air throughout the experiment, although inducing a higher base level of enzyme activities, did not increase the maximum SOD activity obtained relative to control processes gassed continuously with air . The profile of the specific activity of SOD (U mg CDW(-1)) appeared to correlate with dissolved oxygen levels in processes where no H2O2 addition occurred . These findings indicate that it is unsound to use the term "oxidative stress" to encompass a stress response produced by addition of a chemical (H2O2) or by elevated dissolved oxygen levels because the response to each might be quite different. Enzyme Microb Technol, 2000 Nov 15, 27(9), 714 - 723 Scale-up study on suspension cultures of Taxus chinensis cells for production of taxane diterpene; Pan Z et al.; Suspension cells of Taxus chinensis were cultivated in both shake flasks and bioreactors . The production of taxuyunnanine C (TC) was greatly reduced when the cell cultures were transferred from shake flasks to bioreactors . Oxygen supply, shear stress and stripping-off of gaseous metabolites were considered as potential factors affecting the taxane accumulation in bioreactors . The effects of oxygen supply on the cell growth and metabolism were investigated in a stirred tank bioreactor by altering its oxygen transfer rate (OTR) . It was found that both the pattern and amount of TC accumulation were not much changed within the range of OTR as investigated . Comparative studies on the cell cultivation in low shear and high shear generating bioreactors suggest that the decrease of TC formation in bioreactors was not due to the different shear environments in different cultivation vessels . An incorporation of 2% CO(2) in the inlet air was beneficial for the cell growth, but did not improve the TC production in bioreactors . Furthermore, the effects of different levels of ethylene addition into the inlet air on the cell growth and TC production were investigated in a bubble column reactor . The average cell growth rate increased from 0.146 to 0.204 d(-1) as the ethylene concentration was raised from 0 to 50 ppm, and both the content and production of TC were also greatly improved by ethylene addition . At an ethylene concentration of 18 ppm, the highest TC content and volumetric production in the reactor reached 13.28 mg/(g DW) and 163.7 mg/L, respectively, which were almost the same as those in shake flasks . Compared with the control reactor (bubble column without ethylene supplementation), the maximum TC content was increased by 82% and the total production of TC was doubled . The results indicate that ethylene is a key factor in scaling up the process of the suspension cultures of T . chinensis from a shake flask to a bioreactor. Enzyme Microb Technol, 2000 Nov 15, 27(9), 680 - 690 Oxygen transfer and uptake rates during xanthan gum production; Garcia-Ochoa F et al.; Oxygen uptake rate and oxygen mass transfer rate have been studied during xanthan gum production process in stirred tank bioreactor . Empirical equations for the oxygen mass transfer coefficient have been obtained taking into account several variables such as air flow rate, stirrer speed and apparent viscosity . Oxygen uptake rate evolution in the course fermentation has been measured, obtaining an equation as a function of biomass concentration, including overall growth and non growth-associated oxygen uptake . A metabolic kinetic model has been employed for xanthan gum production description including oxygen mass transfer and uptake rates . The results point out that this model is able to describe adequately not only oxygen dissolved evolution, but also of the production of xanthan and substrate consumption . Also, the influence of several parameters (k(L)a, air flow rate and dissolved oxygen) in the evolution of the key compounds of the system have been studied . The results of the simulation shown that an increasing of dissolved oxygen concentration favor the xanthan gum production. Enzyme Microb Technol, 2000 Nov 15, 27(9), 652 - 663 Model-based bioreactor selection for large-scale solid-state cultivation of Coniothyrium minitans spores on oats; Oostraa J et al.; Non-mixed and mixed SSF reactors were evaluated for their applicability in large-scale spore production of the biocontrol fungus Coniothyrium minitans . The major problem to overcome in large-scale SSF is heat accumulation . Testing various cooling strategies in large-scale bioreactors would be very expensive and time consuming, therefore lab experiments in combination with mathematical simulations were used instead . The metabolic heat production rate, estimated from the oxygen consumption rate of C . minitans on oats in Erlenmeyer flasks, was about 500 Watt per m(3) bed . Conductive cooling in packed-bed reactors was insufficient to cool large reactor volumes (radius > 0.2 m) . The poor thermal conductivity of the bed (lambda(b) = 0.1 W m(-2) K(-1)) resulted in steep radial temperature profiles . Adequate temperature control could be achieved with forced aeration, but concomitant water losses lead to significant shrinkage of the oats (30%) and critically low water activities, even though the bed was assumed to be aerated with water saturated air . Mixed systems, however, allowed heat removal without the need of evaporative cooling . Simulations showed that large volumes could be cooled via the wall at low mixing intensities and small temperature driving forces . Experimental studies showed no detrimental effect of mixing on spore production by C . minitans . The spore production yield in a continuously mixed scraped-drum reactor (0.2 rpm) was 5 x 10(12) spores per kg dry oats after 450 hours . Based on the scale-up potential of the mixed system and the absence of detrimental mixing effects it is believed that a mixed bioreactor is superior to a non-mixed system for large-scale production of C . minitans spores. Sheng Wu Gong Cheng Xue Bao, 2000 May, 16(3), 368 - 72 {Studies on simultaneous production of chitosanase and chitosan degradation in situ with Trichoderma reesei in convoluted fibrous bed bioreactor}; Wu MB et al.; The cells of Trichoderma reesei were immobilized on a roll of porous polyurethane foam sheet and packed in a bubble column bioreactor for simultaneous production of chitosanase and degradation of chitosan in situ . The average degree of polymerization could be regulated by reaction time . Under the repeated-batch process with 2% soluble chitosan at pH4.8, 28 degrees C, the activity of chitosanase for each batch was above 0.15 IU/mL, the average yield of reducing sugar as D-glucosamine reached 73% . The novel immobilized bioreactor system run stably and effectively in the successive 10 batches lasting 30 days without notable change in the activity and productivity. Biomaterials, 2000 Dec, 21(23), 2443 - 52 Effects of fluid flow on the in vitro degradation kinetics of biodegradable scaffolds for tissue engineering; Agrawal CM et al.; Scaffolds fabricated from biodegradable polymers are used extensively in the field of tissue engineering . Many of these scaffolds are subjected to fluid flow, either in vivo or in bioreactors ex vivo . The goal of this study was to examine the effects of fluid flow on the degradation characteristics and kinetics of scaffolds in vitro . Scaffolds with different porosity and permeability values were fabricated using a copolymer of polylactic acid and polyglycolic acid . These scaffolds were subjected to degradation in phosphate buffered saline at 37 degrees C for up to 6 weeks under two test conditions: static and flow (250 microl/min) . The porosity of the scaffolds decreased up to 2 weeks and then increased, while the elastic modulus first increased and then decreased over the course of the study . The mass and molecular weight of the scaffolds exhibited a steady decrease up to 6 weeks . The results further indicated that lower the porosity and permeability of the scaffolds, the faster their rate of degradation . Additionally, fluid flow decreased the degradation rate significantly . It is possible that the high rates of degradation observed here were due to autocatalysis of the degradation reaction by the acidic degradation products. Sheng Wu Gong Cheng Xue Bao, 2000 Jul, 16(4), 525 - 7 {On-line measurement of oxygen uptake rate in the cultivation of Vero cells using the dynamic method}; Zhou YJ et al.; The oxygen uptake rate(OUR) during the cultivation of Vero cells in 1.5 L CelliGen bioreactor was on-line determined using the dynamic method . The results showed that the cell growth and metabolic state during the exponential growth phase was lineally related to the OUR . This implies that the on-line measurement of OUR can be used to promptly monitor the physiological state of cultured cells and to efficiently avoid contamination because of frequent sampling in the large-scale cultivation of mammalian cells. Sheng Wu Gong Cheng Xue Bao, 2000 Jul, 16(4), 421 - 4 {Production of pharmaceutical proteins with mammary gland bioreactor}; Liu S et al.; Mammary gland bioreactor is a useful biological system which expresses foreign genes in the mammary gland and produces functional pharmaceutical proteins in milk . This production route is appealing for it's advantages, such as the simplicity of access to the expressed protein, the high production of the mammary gland, the capabilities to perform translational modifications . As an alternative of cell culture systems, it is a new biotechnology . The article reviews some aspects on generation and characterization of mammary gland bioreactor, separation and purification of foreign protein from milk and some questions that need to be answered on the route. Protein Expr Purif, 2000 Nov, 20(2), 324 - 33 Bioreactor-scale production and one-step purification of Epstein-Barr nuclear antigen 1 expressed in baculovirus-infected insect cells; Meij P et al.; Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance . Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes . To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available . This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1) . For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA . We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production . However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells . Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks . Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50% . For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated . Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose . A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95% . Protein Expr Purif, 2000 Nov, 20(2), 228 - 36 Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector; Chen Y et al.; Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A . The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids . To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed . Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography . However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans . Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions . Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing . The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation . These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients . Hum Gene Ther, 2000 Oct 10, 11(15), 2079 - 91 Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo; Gao G et al.; Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy . While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials . The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality . In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system . Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved . We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors . By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications. Biotechnol Bioeng, 2000 Dec 5, 70(5), 553 - 63 Bioconversion of hydrophobic compounds in a continuous closed-gas-loop bioreactor: feasibility assessment and epoxide production; Steinig GH et al.; Microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions . However, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms . This results in low-volumetric productivity in classical bioreactors . The Continuous Closed-Gas-Loop Bioreactor is described here-a reactor system with high productivity, but without the problems associated with two-phase systems, such as an emulsified product stream and phase toxicity . Its working principle is to recirculate a gas phase through a bioreaction compartment and a saturator/absorber module where the product accumulates as a clear organic solution . A wide range of bioconversions should be possible in this set-up, and proof of concept was established for the epoxidation of 1,7-octadiene to (R)-1,2-epoxyoct-7-ene by a native strain of Pseudomonas oleovorans . This reaction represents a group of terminal alkene epoxidations where the bioconversion substrate does not support growth of the microorganism . Practical results at a 5l-scale are presented for this bioconversion for both batch and continuous operation with respect to the aqueous phase, showing continuous stable epoxidation at productivities >14 micromol min(-1) L(-1) (U L(-1)) . The results confirm that the metabolism does not allow a simple optimization strategy, because growth and biotransformation substrates compete for the same enzyme sites, and conversely growth on a substrate using this very enzyme system is necessary for longterm bioconversion . Integrated removal of the CO(2) formed via the liquid overflow was estimated from theory and verified in experimental work . Cancer Biother Radiopharm, 2000 Aug, 15(4), 357 - 66 Growth of tumor-derived activated T cells for the treatment of advanced cancer; Lewko WM et al.; In 1994, we reported on a series of patients treated with T-cell therapy (Study #1) . This paper (Study #2) is an update of our experience through 1999 in the production of tumor-derived activated cells (TDAC), also called tumor-infiltrating lymphocytes (TIL), from tumor biopsies . TDAC were successfully grown in medium containing Interleukin-2 from 75% of the 366 tumor biopsies tested . There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors . Many of the tumors were shipped to the laboratory by overnight delivery from distant sites . Success rate did decrease with the length of time for tumor transport in excess of 24 hours . Certain additional cytokines were tested when cultures did not grow . Interleukin-4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 alone . In order to produce TDAC to treat patients, cells were grown in gas permeable plastic bags or in artificial capillary bioreactor cultures . Approximately 1 x 10(9) were seeded from an initially successful "feasibility study" to bulk produce cells for treatment . Harvest was carried out after about 3 weeks . Sixty-three patients were treated at least once with a minimum of 1 x 10(10) TDAC given by intravenous infusion . On the average, the number of cells per treatment was 3 x 10(10) with a viability of 87% . TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells . Secreted granulocyte-monocyte colony stimulating factor, interferon gamma and tumor necrosis factor alpha were measured in TDAC conditioned medium . Only 34 patients received the full course of 4 TDAC treatments . The cells were well tolerated with mild fever and dyspnea . Partial responses were observed in 8 patients, including the dramatic regression of scalp nodules in a patient with renal cancer . These results showed that therapeutic amounts of TDAC can be produced in cell culture in a reasonable and cost-effective manner . The cells were well tolerated and responses were seen in renal and melanoma patients resistant to IL-2 with bulky, advanced cancer. Bratisl Lek Listy, 2000, 101(6), 340 - 7 {The niches and pathways of animal pathogens}; Mikula I et al.; Infectious diseases are not a relict of the past but a topical phenomenon determined by complex evolution of the currently existing and constantly changing microbial agents and their hosts . With regard to abundance of species within the microbial kingdom and rate of its changes and development, it is difficult to predict the role of the microbial factor in mortality of humans and animals . The study and generalization of sequential similarities of microbial virulence factors after the completion of genome sequencing of principal pathogens can play a positive role in this direction . At present, molecular-genetic methods allow us to study the phylogenetic relationships of microbes and categorize them according to new criteria . The efficient control of diseases caused by microbes requires knowledge on their physiological and ecological niche from which they penetrate, in various ways, into the host organisms and, under suitable conditions, induce mass diseases . This process has several stages and, in the recent period, it is increasingly affected by human activities . The knowledge on all participants in this process, i.e . the microbe and its niche, factors of virulence and pathways of their dissemination, requires a scientifically based surveillance . Abundance and variability is characteristic for both microbial kingdom and microbial niche . Some identification of pathogenic properties of microorganisms and factors affecting their movement from their niche to the recipients results in activation of old classical diseases (e.g . plague, cholera, tuberculosis etc.) or emerging of new, so far unknown infections diseases ("emerging inf . disease"--EID), caused for example by lentiviruses, oncoviruses, filiviruses, bartonella, borrelia etc . This has provided the basis for establishment of new medical trends and approaches, such as "Emergency medicine" or "Travel medicine", expressing their purpose by their names . The control of existing or proposed infectious diseases in the 21st century (in which majority of factors such as urbanization, environmental factors, evolution of the microbial kingdom, will contribute to the persistence or "emergence" of new diseases) will be affected by the input of new knowledge in the field of molecular biology, such as introduction of biosensors, genetic tests, microchips, new generation of DNA vaccines, enteric vaccines and antibodies produced by transgenic animal bioreactors or plants, "customized" vaccines assessed for individual genetic profiles, etc . (Tab . 5, Ref . 21.) Chin J Biotechnol, 1999, 15(4), 239 - 44 Study on rt-PA production by recombinant CHO cells immobilized with a porous microcarrier; Zhaolie C et al.; Using the porous microcarrier Cytopore, immobilized cultivation of a recombinant CHO cell line 4B3 producing tissue type plasminogen activator (rt-PA) was performed in a 3L stirring tank bioreactor with the perfusion of serum-free medium DF5S . 4B3 cells could enter the inner space and grow both in the inner space and on the surface of the Cytopore . The highest viable cell density of 4B3 and the highest production level of rt-PA were 8.83 x 10(6) cells/mL and 12,473 IU/mL, respectively . Purified by MPG adsorption chromatography and lysine-Sepharose 4B affinity chromatography, the purified rt-PA with a purity exceeding 98% was obtained from the culture supernatant. Adv Biochem Eng Biotechnol, 2000, 68, 191 - 233 Recovery of proteins and microorganisms from cultivation media by foam flotation; Schugerl K; Foaming is often present in aerated bioreactors . It is undesired, because it removes the cells and the cultivation medium from the reactor and blocks the sterile filter . However, it can be used for the recovery of proteins and microorganisms from the cultivation medium . The present review deals with the characterization of model protein foams and foams of various cultivation media . The suppression of foaming by antifoam agents and their effect on the oxygen transfer rate, microbial cell growth and product formation are discussed . The influence of process variables on the recovery of proteins by flotation without and with surfactants and mathematical models for protein flotation are presented . The effect of cultivation conditions, flotation equipment and operational parameters on foam flotation of microorganisms is reviewed . Floatable and non-floatable microorganisms are characterized by their surface envelope properties . A mathematical model for cell recovery by flotation is presented . Possible application areas of cell recovery by flotation are discussed. Adv Biochem Eng Biotechnol, 2000, 68, 61 - 138 Biochemical engineering aspects of solid state bioprocessing; Mitchell DA et al.; Despite centuries of use and renewed interest over the last 20 years in solid-state fermentation (SSF) technology, and despite its good potential for a range of products, there are currently relatively few large-scale commercial applications . This situation can be attributed to the complexity of the system: Macroscale and microscale heat and mass transfer limitations are intrinsic to the system, and it is only over the last decade or so that we have begun to understand them . This review presents the current state of understanding of biochemical engineering aspects of SSF processing, including not only the fermentation itself, but also the auxiliary steps of substrate and inoculum preparation and downstream processing and waste disposal . The fermentation step has received most research attention . Significant advances have been made over the last decade in understanding how the performance of SSF bioreactors can be controlled either by the intraparticle processes of enzyme and oxygen diffusion or by the macroscale heat transfer processes of conduction, convection, and evaporation . Mathematical modeling has played an important role in suggesting how SSF bioreactors should be designed and operated . However, these models have been developed on the basis of laboratory-scale data and there is an urgent need to test these models with data obtained in large-scale bioreactors. J Biotechnol, 2001 Nov 17, 84(1), 13 - 25 Estimation of kinetic rates in batch Thiobacillus ferrooxidans cultures; Biagiola S et al.; In this work, the key problem of estimation in bioprocesses when no structural model is available is dealt with . A nonlinear observer-based algorithm is developed in order to estimate kinetic rates in batch bioreactors . The algorithm uses the measurements of biomass concentration and either substrate concentration or redox potential to perform the estimation of the respective specific kinetic rates . For this purpose, a general mathematical model description of the process is provided . The estimation algorithm design is based on a nonlinear reduced-order observer . The observer performance is validated with experimental results on a Thiobacillus ferrooxidans batch culture. J Biotechnol, 2001 Nov 17, 84(1), 1 - 12 Continuous enantioselective esterification of trans-2-phenyl-1-cyclohexanol using a new Candida rugosa lipase in a packed bed bioreactor; Sanchez A et al.; Enantioselective resolution of trans-2-phenyl-1-cyclohexanol (TPCH) by a Candida rugosa lipase, obtained by fermentation in the laboratory, and immobilised on EP100 polypropylene powder has been carried out using isooctane as solvent and propionic acid as esterifying agent . The study have included the utilisation of this biocatalyst in a batch process and the optimisation of the esterification conditions by means of a Box-Hunter-based experimental design . The main variables controlling the process, concentration of acid and alcohol, have been numerically optimised using initial esterification rate as objective function . The optimal concentrations for the batch process were 50 mM for the alcohol and 71 mM for the acid . This esterification reaction kinetics corresponded to a reversible Michaelis-Menten kinetic law for the optimal conditions, which has permitted to select a plug-flow packed bed bioreactor as the most appropriate configuration to minimise the residence time and to avoid shear stress effect on the biocatalyst . The behaviour of the continuous packed bed bioreactor at two different residence times (302 and 582 min) was in accordance with predictions from batch experiments, with slightly deviations (less than 10%) . Continuous experiments maintained high values of enantioselectivity (enantiomeric factor was practically 1) and conversion near equilibrium value (35%) when long-time operation was carried out . Besides, long-time stability of biocatalyst has permitted to scale-up the production of enantioenriched (1R,2S)-TPCH propionate to yield gram quantities. Biotechnol Prog, 2000 Sep-Oct, 16(5), 905 - 16 Computational model for effects of ligand/receptor binding properties on interleukin-2 trafficking dynamics and T cell proliferation response; Fallon EM et al.; Multisubunit cytokine receptors such as the heterotrimeric receptor for interleukin-2 (IL-2) are ubiquitous in hematopoeitic cell types of importance in biotechnology and are crucial regulators of cell proliferation and differentiation behavior . Dynamics of cytokine/receptor endocytic trafficking can significantly impact cell responses through effects of receptor down-regulation and ligand depletion, and in turn are governed by ligand/receptor binding properties . We describe here a computational model for trafficking dynamics of the IL-2 receptor (IL-2R) system, which is able to predict T cell proliferation responses to IL-2 . This model comprises kinetic equations describing binding, internalization, and postendocytic sorting of IL-2 and IL-2R, including an experimentally derived dependence of cell proliferation rate on these properties . Computational results from this model predict that IL-2 depletion can be reduced by decreasing its binding affinity for the IL-2R betagamma subunit relative to the alpha subunit at endosomal pH, as a result of enhanced ligand sorting to recycling vis-a-vis degradation, and that an IL-2 analogue with such altered binding properties should exhibit increased potency for stimulating the T cell proliferation response . These results are in agreement with our recent experimental findings for the IL-2 analogue termed 2D1 {Fallon, E . M . et al . J . Biol . Chem . 2000, 275, 6790-6797} . Thus, this type of model may enable prediction of beneficial cytokine/receptor binding properties to aid development of molecular design criteria for improvements in applications such as in vivo cytokine therapies and in vitro hematopoietic cell bioreactors. Biotechnol Prog, 2000 Sep-Oct, 16(5), 893 - 6 Comparison of chondrogensis in static and perfused bioreactor culture; Pazzano D et al.; As a result of the low yield of cartilage from primary patient harvests and a high demand for autologous cartilage for reconstructive surgery and structural repair, primary explant cartilage must be augmented by tissue engineering techniques . In this study, chondrocytes seeded on PLLA/PGA scaffolds in static culture and a direct perfusion bioreactor were biochemically and histologically analyzed to determine the effects of fluid flow and media pH on matrix assembly . A gradual media pH change was maintained in the bioreactor within 7.4-6.96 over 2 weeks compared to a more rapid decrease from 7.4 to 6.58 in static culture over 3 days . Seeded scaffolds sub