Bioreactor

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 subjected to 1 microm/s flow demonstrated a 118% increase (p < 0.05) in DNA content, a 184% increase (p < 0.05) in GAG content, and a 155% (p < 0.05) increase in hydroxyproline content compared to static culture . Distinct differences were noted in tissue morphology, including more intense staining for proteoglycans by safranin-O and alignment of cells in the direction of media flow . Culture of chondrocyte seeded matrices thus offers the possibility of rapid in vitro expansion of donor cartilage for the repair of structural defects, tracheal injury, and vascularized tissue damage.

Biotechnol Prog, 2000 Sep-Oct, 16(5), 872 - 84
293SF metabolic flux analysis during cell growth and infection with an adenoviral vector; Nadeau I et al.; Metabolic flux quantification of cell culture is becoming a crucial means to improve cell growth as well as protein and vector productions . The technique allows rapid determination of cell culture status, thus providing a tool for further feeding improvements . Herein, we report on key results of a metabolic investigation using 293 cells adapted to suspension and serum-free medium (293SF) during growth and infection with an adenoviral vector encoding the green fluorescence protein (GFP) . The model developed contains 35 fluxes, which include the main fluxes of glycolysis, glutaminolysis, and amino acids pathways . It requires specific consumption and production rate measurements of amino acids, glucose, lactate, NH(3), and O(2), as well as DNA and total proteins biosynthesis rate measurements . Also, it was found that extracellular protein concentration measurement is important for flux calculation accuracy . With this model, we are able to describe the 293SF cell metabolism, grown under different culture conditions in a 3-L controlled bioreactor for batch and fed-batch with low glucose . The metabolism is also investigated during infection under two different feeding strategies: a fed-batch starting at the end of the growth phase and extending during infection without medium change and a fed-batch after infection following medium renewal . Differences in metabolism are observed between growth and infection, as well as between the different feeding strategies, thus providing a better understanding of the general metabolism.

Biotechnol Prog, 2000 Sep-Oct, 16(5), 822 - 8
First steps in robot automation of sampling and sample management during cultivation of mammalian cells in pilot scale; Lutkemeyer D et al.; The robot automation of sampling and the subsequent treatment and storage of aliquots during mammalian cell cultivations was investigated . The complete setup, the development and testing of the sampling device, the robot arm, and the cell imaging system are described . The developed sampling device is directly coupled to a pilot bioreactor . It allows the computerized sterile filling of cell broth into 50 mL sample tubes . After each sampling the whole tubing system is steam sterilized . For further off-line treatment a robot takes the sample to the different devices . This robot is equipped with a camera and a force/torque sensor . A color-based object recognition guides the arm in a complex surrounding with different illumination situations, enabling the robot to load the sampling device with tubes and take the sample to further devices . For necessary pipetting and refilling we developed a computerized device . Cells are automatically stained and counted using an imaging system . The cell number and viability is automatically saved in a process control system together with the on-line parameters . During several cultivations in 20 and 100 L scale these main components of the automation strategy were successfully tested.

Biotechnol Prog, 2000 Sep-Oct, 16(5), 744 - 50
Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system; Hu YC et al.; The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications . This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates . A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues . In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus . During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase . The chosen production method was based on using the packed-bed bioreactor . The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor . The process included a growth stage followed by a production stage . In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses . The expressed protein was secreted, collected from the culture fluid, and purified . The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers.

Biotechnol Prog, 2000 Sep-Oct, 16(5), 703 - 9
Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor; Stiens LR et al.; For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts . Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts . Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor . Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1) . Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium . The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days . The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity . The obtained receptor showed improved TSH binding . The process development was based on cultivations in a 2-L bench-scale bioreactor . Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared . After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor . The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.

Enzyme Microb Technol, 2000 Nov 1, 27(8), 631 - 635
Increase in Chlorella strains calorific values when grown in low nitrogen medium; Illman AM et al.; The calorific value of five strains of Chlorella grown in Watanabe and low-nitrogen medium was determined . The algae were grown in small (2L) stirred tank bioreactors and the best growth was obtained with Chlorella vulgaris with a growth rate of 0.99 d(-1) and the highest calorific value (29 KJ/g) was obtained with C . emersonii . The cellular components were assayed at the end of the growth period and the calorific value appears to be linked to the lipid content rather than any other component.

Bioelectromagnetics, 2000 Oct, 21(7), 491 - 8
Effect of low frequency, low amplitude magnetic fields on the permeability of cationic liposomes entrapping carbonic anhydrase: I . Evidence for charged lipid involvement; Ramundo-Orlando A et al.; The influence of low frequency (4-16 Hz), low amplitude (25-75 mu T) magnetic fields on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied . Cationic liposomes containing dipalmitoylphosphatidylcholine, cholesterol, and charged lipid stearylamine (SA) at different molar ratios (6:3:1 or 5:3:2) were used . Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (p-NPA) across intact liposome bilayer . After 60 min of exposure to 7 Hz sinusoidal (50 mu T peak) and parallel static (50 mu T) magnetic fields the enzyme activity, as a function of increased diffusion rate of p-NPA, rose from 17 +/- 3% to 80 +/- 9% (P < .0005, n = 15) in the 5:3:2 liposomes . This effect was dependent on the SA concentration in the liposomes . Only the presence of combined sinusoidal (AC) and static (DC) magnetic fields affected the p-NPA diffusion rates . No enzyme leakage was observed . Such studies suggest a plausible link between the action of extremely low frequency magnetic field on charged lipids and a change of membrane permeability .

Ann Ital Chir, 2000 May-Jun, 71(3), 337 - 45
{In vitro experimentation of a new model of radial flow bioreactor containing isolated hepatocytes}; Morsiani E et al.; Hepatocyte based artificial liver support systems are under investigation to support acute liver failure patients . The main purpose of such systems is to serve as a bridge to liver transplantation, or to promote spontaneous liver recovery . Limitation in mass-transfer capacity makes hollow-fiber bioreactors unsuited for long-term functioning of hybrid devices . We developed a novel radial-flow bioreactor in which the fluid perfuses the module from the center to the periphery, after having diffused through a space occupied by a three-dimensional structure filled with the hepatocytes . Five grams of freshly isolated porcine hepatocytes were seeded into uncoated, woven-non woven, hydrophilic polyester fabric, overlaid by two polyethersulfone membranes . Liver cells were perfused with 37 degrees C-warm, oxygenated, serum-free tissue culture medium, in which NH4Cl and Lidocaine were added at the final concentration of 1 mM and 60 micrograms/ml, respectively . Ammonium chloride removal, urea synthesis, monoethylglycinexylide (MEGX), pO2, pCO2, and pH were measured throughout the 14 day duration of the study . In a separate set of experiments, a scaled-up version of the radial flow bioreactor containing 150 grams of cells was perfused for 7 h with recirculating human plasma and MEGX production was monitored . During the 2 weeks of the study, an increasing production of urea was paralleled by constant ammonium removal . MEGX concentration after Lidocaine addition increased throughout the 14 days of perfusion with tissue culture medium, as well as after 7 hour perfusion with human plasma . Under transmission and scanning electron microscopy cells appeared attached to the polyester and one to each other, displaying ultrastructural features typical of functioning hepatocytes . Our study showed that liver cells were metabolically active when perfused into the radial-flow bioreactor . This configuration allowed close contact between media, or plasma, and cells at a physiological flow rate, by equalizing the concentration of the perfusing components, including O2, throughout the module . Our results suggest a potential use of this system for temporary extracorporeal liver support in acute hepatic failure patients.

Ann Ital Chir, 2000 May-Jun, 71(3), 311 - 8
{Hybrid (bioartificial) systems for liver support . History and current developments}; Morsiani E; Attempts to develop liver support systems for the treatment of acute liver failure patients have ranged in the past, from the use of hemodialysis, or plasma exchange, or activated charcoal particles and synthetic resins, as well as the use of bioreactors loaded with liver tissue . However, no system demonstrated a significant improvement of patient survival, nor has achieved a wide clinical use . Liver transplantation remains the only treatment for severe hepatic failure that can improve patient survival . On the other hands, the chronic scarcity of organs for transplantation, leads to an urgent necessity of liver support systems . In this paper, we reviewed the historical experience and current status of artificial liver support systems, with particular emphasis on the so-called hybrid or bioartificial liver, in which to the traditional artificial components, such as selective membranes, charcoal particles and resins, isolated hepatocytes are used.

Biotechnol Bioeng, 2000 Nov 20, 70(4), 446 - 55
Error-prone PCR of Vitreoscilla hemoglobin (VHb) to support the growth of microaerobic Escherichia coli; Andersson CI et al.; Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions . It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used error-prone PCR to generate a number of randomly mutated vhb genes to be expressed and studied in Escherichia coli . In addition, the mutated VHb proteins also contained an extension of eight residues (MTMITPSF) at the amino terminus . VHb mutants were screened for improved growth properties under microaerobic conditions and 15 clones expressing mutated hemoglobin protein were selected for further characterization and cultivated in a microaerobic bioreactor to analyze the physiological effects of novel VHb proteins on cell growth . The expression of four VHb mutants, carried by pVM20, pVM50, pVM104, and pVM134, were able to enhance microaerobic growth of E . coli by approximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium . The vhb gene in pVM20 contains two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly . pVM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66 . pVM104 and pVM134 express VHb proteins carrying the mutations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively . Our experiments also indicate that the positive effects elicited by mutant VHb-expression from pVM20 and pVM50 are linked to the peptide tail . Removal of the N-terminal sequence reduced cell growth approximately 23% and 53%, respectively, relative to wild-type controls . These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics for improving microaerobic growth of E . coli by using combined mutation techniques, addition of a peptide tail, and random error-prone PCR .

Biotechnol Appl Biochem, 2000 Oct, 32 ( Pt 2), 127 - 35
High-level synthesis of human prolactin in Chinese-Hamster ovary cells; Soares CR et al.; Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells . One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation . The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA . Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/10(6) cells per day, could be rapidly obtained; production by pEDdc-hPRL-transfected cells was about 10-fold lower . However, a three-step MTX amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day . A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day . SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL . Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay) . This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.

Plant Mol Biol, 2000 Jun, 43(2-3), 347 - 59
Plants as bioreactors for protein production: avoiding the problem of transgene silencing; De Wilde C et al.; Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures . The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations . Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories . Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level . Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.

Trends Biotechnol, 2000 Oct, 18(10), 420 - 32
Animal-cell damage in sparged bioreactors; Chisti Y; The gas sparging of culture broth causes damage to suspended animal cells . However, despite this, sparged bioreactors remain the preferred means of cell culture because sparging is a robust method of supplying oxygen, especially on a large scale . This article examines the underlying mechanisms involved in bubble-associated cell damage and the methods available for controlling such damage.

Tissue Eng, 2000 Aug, 6(4), 297 - 305
Combining chondrocytes and smooth muscle cells to engineer hybrid soft tissue constructs; Brown AN et al.; Engineering new tissues using cell transplantation may provide a valuable tool for reconstructive surgery applications . Chondrocyte transplantation in particular has been successfully used to engineer new tissue masses due to the low metabolic requirements of these cells . However, the engineered cartilaginous tissue is too rigid for many soft tissue applications . We propose that hybrid tissue engineered from chondrocytes and smooth muscle cells could reflect mechanical properties intermediate between these two cell types . In this study, rat aortic smooth muscle cells and pig auricular chondrocytes were co-cultured on polyglycolic acid fiber-based matrices to address this hypothesis . Mixed cell suspensions were seeded by agitating the polymer matrices and a cell suspension with an orbital shaker . After seeding, cell-polymer constructs were cultured in stirred bioreactors for 8 weeks . The cell density and extracellular matrix (collagen, elastin, and glycosaminoglycan) content of the engineered tissues were determined biochemically . After 8 weeks in culture, the hybrid tissue had a high cell density (5.8 x 108 cells/cm(3)), and elastin (519 microg/g wet tissue sample), collagen (272 microg/g wet tissue sample), and glycosaminoglycan (GAG; 10 microg/g wet tissue sample) content . Mechanical testing indicated the compressive modulus of the hybrid tissues after 8 weeks to be 40.8 +/- 4.1 kPa and the equilibrium compressive modulus to be 8.4 +/- 0.8 kPa . Thus, these hybrid tissues exhibited intermediate stiffness; they were less stiff than native cartilage but stiffer than native smooth muscle tissue . This tissue engineering approach may be useful to engineer tissues for a variety of reconstructive surgery applications.

Biotechnol Bioeng, 2000 Nov 5, 70(3), 247 - 53
Rheological properties and mechanical stability of new gel-entrapment systems applied in bioreactors; Vogelsang C et al.; The mechanical stability of gels applied for entrapment and retention of biocatalysts in bioreactors is of crucial importance for successful scale-up applications . Gel abrasion in agitated reactors will depend on liquid shear, bubble shear, and wall shear, as well as collisions between the gel particles . As a simplified standardized model system, abrasion of gel beads was studied in 1-m-high bubble columns with controlled aeration, and quantified by measuring the loss of gel material into solution . Gel beads were also taken out to measure stress-strain response during controlled compression . More general rheological properties of different gels were studied by applying a variety of regimes of controlled compression of standardized gel cylinders: Gel strength was measured by recording the fracture properties and the Young's modulus . Viscoelastic properties were revealed by recording creep during compression as well as recovery after compression . Oscillation tests up to 1000 cyclic compressions were applied to compare the fatigue of different gels . Results obtained for Ca-alginate gels, gels of chemically modified polyvinyl alcohol with stilbazolium groups (PVA-SbQ) as well as mixed gels of Ca-alginate and PVA-SbQ are compared with previously published data for kappa-carrageenan, agar, and polyethylene glycol (PEG) gels . It is concluded that material fatigue rather than mechanical properties such as stiffness or fracture stress should be considered when selecting a suitable gel material on the basis of abrasion resistance . The very soft and superelastic PVA-SbQ gel showed no significant fatigue in mechanical tests and no abrasion was detected in the standardized model system used . Ca-alginate gels, however, showed severe irreversible changes due to fatigue at oscillating loads and creep at constant load . Due to their similarities with kappa-carrageenan gels in mechanical tests, it is likely that Ca-alginate would also be sensitive to abrasion . Mixed gels of Ca-alginate and PVA-SbQ represent a complex system with intermediate properties, showing significant fatigue and creep, but elastic properties from the PVA-SbQ gel make it less sensitive than the pure Ca-alginate gel .

J Biotechnol, 2000 Aug 25, 81(2-3), 95 - 105
High level benzodiazepine and ammonia clearance by flat membrane bioreactors with porcine liver cells; Bader A et al.; The onset of hepatic encephalopathy is a multifactorial process in which endogenous benzodiazepines and hyperammonemia play a pivotal role . The treatment of comatose states in liver failure is one of the major functions of a bioartificial liver . A controlled study demonstrating the capacity of a large scale bioartificial liver to detoxify benzodiazepines could be a crucial prerequisite to break this circle of events leading to coma . The aim of this study was therefore to expose the bioreactor to high levels of benzodiazepines and ammonia for evaluation of its detoxifying capacity . We have developed a novel and unique device reconstructing the plate architecture of the liver . Porcine hepatocytes were co-cultured with non-parenchymal cells . We investigated benzodiazepine metabolism using diazepam as model drug . The bioreactor was also loaded with high levels of ammonia and ammonia clearance as well as urea secretion with ammonia challenge were investigated . Albumin secretion was analysed in parallel as a control viability and tissue specific secretory parameter . The results clearly show that the velocity of diazepam turnover increases between day 1 and 2 and stabilises at high levels . Typical diazepam metabolites including temazepam, N-desmethyl-diazepam and oxazepam were generated . Cell specific functions, including albumin secretion, were comparable to an in vivo liver . We conclude that the flat membrane bioreactor used as bioartificial liver has the potential to detoxify diazepam and ammonia at significant amounts . Maintenance of monoxygenase activities in vitro is one of the strongholds of the bioreactor concept presented in this study.

Mol Biotechnol, 2000 Jul, 15(3), 249 - 57
Adaptation of mammalian cells to growth in serum-free media; Sinacore MS et al.; A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture . The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein . The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium . The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives . The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells . Examples are presented for the use of this protocol for recombinant CHO cells.

Enzyme Microb Technol, 2000 Oct 1, 27(7), 502 - 510
The Vancomycin biosynthetic pathway is induced in oxygen-limited Amycolatopsis orientalis (ATCC 19795) cultures that do not produce antibiotic; Dunstan GH et al.; Vancomycin biosynthetic capacity was stimulated in oxygen-limited bioreactor culture (6.4% DOT) of Amycolatopsis orientalis, coinciding with a down-regulation of the culture growth and protein synthesis rates . However no Vancomycin was detected . ATP production was similar in oxygen-limited and oxygen-sufficient cultures (400-150 micromole(ATP) . g(biomass)(-1)) . Our findings suggest a critical requirement for oxygen flux through the Vancomycin biosynthetic pathway, which is only met under nutrient-limited conditions in which oxygen is in excess.

Magn Reson Med, 2000 Sep, 44(3), 367 - 72
31P NMR spectroscopy of developing cartilage produced from chick chondrocytes in a hollow-fiber bioreactor; Petersen EF et al.; 31P NMR was used to measure the concentrations and spin-lattice relaxation times of phosphorus-containing metabolites in neocartilage developing in an NMR-compatible hollow-fiber bioreactor over four weeks . Separate studies were performed for tissue developing from chondrocytes taken from the proximal and the distal sternum of the chick embryo . The metabolite ratio beta-ATP/Pi did not change significantly with development (proximal: beta-ATP/Pi = 0.38+/- 0.12 at one week, beta-ATP/Pi = 0.44+/-0.07 at four weeks, P< 0.63; distal: beta-ATP/Pi = 0.39+/-0.05 at one week, beta-ATP/Pi = 0.66+/- 0.26 at four weeks, P<0.28) . ATP spin-lattice relaxation times were found to be comparable to those in muscle and brain tissue (proximal: T(1)(beta-ATP) = 0.5+/-0.06 sec at one week, T(1)(beta-ATP) = 0.4+/- 0.01 sec at four weeks; distal: T(1)(beta-ATP) = 0.3+/-0.12 sec at one week, T(1)(beta-ATP) = 0.4+/-0.04 sec at four weeks) . A large increase in the spin-lattice relaxation time of inorganic phosphate, from 1.2+/-0.13 sec to 3.8+/-0.04 sec (P<0.0001) over four weeks of growth, was observed in tissue developing from chondrocytes harvested from the proximal sternum . No comparable increase in T(1)(Pi) was found in tissue developing from chondrocytes harvested from the distal portion of the sternum, which ossifies later in vivo.

Biotechnol Bioeng, 2000 Oct 20, 70(2), 176 - 86
Inhibitory role of root hairs on transport within root culture bioreactors; Bordonaro JL et al.; An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs . Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture . These root cultures were used to demonstrate decreased flow resistance in a tubular reactor as a result of root hair removal . To assess the impact on bioreactor performance, hairy and hairless root cultures were grown in a highly characterized 15-L bubble column bioreactor . In the absence of root hairs, the mixing was greatly enhanced; mixing times became shorter for the hairless culture at roughly 100 g (fresh weight)/L . By the end of the 3-week culture period, the mixing time of the hairy culture was 29 times longer than that of the hairless culture . The growth rate of the hairless culture in the bioreactor was as much as 2.4 times greater than growth of the hairy culture under the same conditions . The improved reactor performance was reflected in greater biomass accumulation and respiratory activity . These results show that the root hairs-which facilitate nutrient uptake in a static soil environment-are detrimental to growth in a liquid environment as an effect of their stagnating fluid flow and limiting oxygen availability .

Biotechnol Bioeng, 2000 Oct 20, 70(2), 160 - 6
Polyurethane membrane as an efficient immobilization carrier for high-density culture of rat hepatocytes in the fixed-bed reactor; Kurosawa H et al.; A fixed-bed bioreactor with a polyurethane membrane (PUM) as a cell-supporting material was developed for high-density culture of rat hepatocytes . The PUM has a heterogeneous porous structure of micropores (pore size <100 microm) and macropores (pore size >100 microm) with a porosity of 90% . One important feature of a PUM is that the macropores have finger-like structures and their diameters gradually decrease from the upper to the lower layer of the PUM . Most rat hepatocytes were readily immobilized in the micropores of PUM . Immobilized cell densities of 1-3 x 10(7) cells/cm(3) PUM were achieved within 5 min by natural downflow of cell suspension and their immobilization efficiencies were more than 99% . Using a syringe pump, a cell density of 5 x 10(7) cells/cm(3) PUM was achieved with more than 96% immobilization efficiency . Perfusion cultures using this reactor were performed for 7 days without cell leakage . The optimal cell density for albumin secretion was between 2 x 10(7) and 3 x 10(7) cells/cm(3) PUM . Albumin secretion in the perfusion culture was maintained for a relatively long period of time when compared to that in the monolayer culture . The rate of albumin secretion in the perfusion culture was about 50% of that in monolayer culture . Hepatocytes immobilized in PUM were slightly aggregated, but they maintained spherical form individually even after 7 days of cultivation . The above results show that PUM is a promising cell-supporting material for efficient immobilization of high cell density of hepatocytes .

Biotechnol Bioeng, 2000 Oct 20, 70(2), 117 - 30
Computational and experimental investigation of flow and fluid mixing in the roller bottle bioreactor; Unger DR et al.; The fully three-dimensional velocity field in a roller bottle bioreactor is simulated for two systems (creeping flow and inertial flow conditions) using a control volume-finite element method, and validated experimentally using particle imaging velocimetry . The velocity fields and flow patterns are described in detail using velocity contour plots and tracer particle pathline computations . Bulk fluid mixing in the roller bottle is then examined using a computational fluid tracer program and flow visualization experiments . It is shown that the velocity fields and flow patterns are substantially different for each of these flow cases . For creeping flow conditions the flow streamlines consist of symmetric, closed three-dimensional loops; and for inertial flow conditions, streamlines consist of asymmetric toroidal surfaces . Fluid tracers remain trapped on these streamlines and are unable to contact other regions of the flow domain . As a result, fluid mixing is greatly hindered, especially in the axial direction . The lack of efficient axial mixing is verified computationally and experimentally . Such mixing limitations, however, are readily overcome by introducing a small-amplitude vertical rocking motion that disrupts both symmetry and recirculation, leading to much faster and complete axial mixing . The frequency of such motion is shown to have a significant effect on mixing rate, which is a critical parameter in the overall performance of roller bottles .

Cells Tissues Organs, 2000, 167(2-3), 171 - 83
Simulated microgravity and hypergravity attenuate heart tissue development in explant culture; Lwigale PY et al.; Exposure to altered gravity may disturb the cytoskeleton-cell surface-extracellular matrix (ECM) interface of embryonic cells . Development of organs such as the heart depends on dynamic interactions across cell surfaces . Fibronectin (FN), for example, a glycoprotein that links the ECM to the cytoskeleton through integrin surface receptors, is required for normal heart development . Thus, altered gravity may perturb organogenesis . We cultured precardiac explants from chick embryos in a rotating bioreactor vessel to simulate microgravity (microG), or in a tissue culture centrifuge, for 18 h during heart development . Bioreactor microG did not alter external morphology of explants, but did significantly reduce the proportion that developed contractions . Immunostaining for FN of explant sections showed that it also significantly reduced the linear extent of staining present in basement membrane regions . Analysis of ultrastructure revealed a significant reduction in the number of desmosomes per unit area and other differences . Hypergravity dramatically abolished development of contractions and altered morphogenesis . The results indicate a probable sensitivity of cardiomyogenic development involving FN to altered gravity .

J Magn Reson, 2000 Sep, 146(1), 49 - 57
In vitro monitoring of total choline levels in a bioartificial pancreas: (1)H NMR spectroscopic studies of the effects of oxygen level; Long RC Jr et al.; This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of (1)H NMR detectable metabolites from alginate/poly-l-lysine/alginate (APA)-encapsulated betaTC3 cells . Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days . During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h . The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation . The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P<0.01) . The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption . A step-up in oxygen level led to a new, higher value for the total choline to reference ratio . From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine . Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine . Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested . In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed .

Appl Environ Microbiol, 2000 Sep, 66(9), 4050 - 7
Parallel processing of substrate correlates with greater functional stability in methanogenic bioreactor communities perturbed by glucose; Hashsham SA et al.; Parallel processing is more stable than serial processing in many areas that employ interconnected activities . This hypothesis was tested for microbial community function using two quadruplicate sets of methanogenic communities, each set having substantially different populations . The two communities were maintained at a mean cell residence time of 16 days and a mean glucose loading rate of 0.34 g/liter-day in variable-volume reactors . To test stability to perturbation, they were subjected to an instantaneous glucose pulse that resulted in a 6.8-g/liter reactor concentration . The pattern of accumulated products in response to the perturbation was analyzed for various measures of functional stability, including resistance, resilience, and reactivity for each product . A new stability parameter, "moment of amplification envelope," was used to compare the soluble compound stability . These parameters indicated that the communities with predominantly parallel substrate processing were functionally more stable in response to the perturbation than the communities with predominantly serial substrate processing . The data also indicated that there was good replication of function under perturbed conditions; the degrees of replication were 0.79 and 0.83 for the two test communities.

Appl Environ Microbiol, 2000 Sep, 66(9), 4037 - 44
Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase; Sabra W et al.; The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate . In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A . vinelandii grown in phosphate-limited nitrogen-free chemostat culture . Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture . Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly . Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A . vinelandii . The composition, thickness, and compactness of this alginate capsule also varied significantly . In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content . The alginate capsule was accordingly thicker and more compact . In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor . Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell . It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.

Matrix Biol, 2000 Aug, 19(4), 353 - 7
Tissue engineering of vascular grafts; Ratcliffe A; The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense . Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable . For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells . A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength . Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo . In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels . This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks . These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.

Biochem Soc Trans, 2000, 28(4), 350 - 3
Recombinant expression systems for the production of collagen; Bulleid NJ et al.; The ability of triple-helical collagen molecules to assemble into supramolecular structures forms the basis of commercial uses of collagen in the food industry and in medical applications such as cosmetic surgery and tissue repair . We have used cDNA techniques to engineer novel collagens with potentially enhanced biological properties; however, expression of fully functional novel molecules is difficult due to the complex nature of procollagen biosynthesis . This article outlines the application of various expression systems to procollagen production and details the use of the mammary gland as a suitable bioreactor for the synthesis of significant amounts of novel procollagens from cDNA constructs.

Biochem, Eng . J. . 2000 Oct 1, 6(2), 109 - 144
Multiple-impeller systems with a special emphasis on bioreactors: a critical review; Gogate PR et al.; The multiple-impeller agitated systems are compared with single-impeller agitated systems with a special focus on its applications for bioreactors . Correlations reported in the literature for gas phase hold-up, mass transfer coefficient and power consumption under gassed and ungassed conditions are compared and recommendations have been made regarding their suitability for design and scale-up of bioreactors . The multiple-impeller systems are found to be superior as compared to single-impeller systems in all the above mentioned aspects, except liquid mixing . For all kinds of reactors where the sole purpose is mass transfer, multiple-impeller systems are advantageous and there would be large savings on an industrial scale, especially for the bioreactors where the reaction periods are long and the power consumption cost could be a significant component to the overall production costs.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 33 - 6
Growth of Steptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production; Fang A et al.; Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production . With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited . Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 9 - 13
Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures; Strauch O et al.; The entomopathogenic nematode-bacterium complex Heterorhabditis megidis Photorhabdus luminescens was cultured in 10-1 internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm . Process parameters like impeller velocity and oxygen saturation were controlled at equal set points . The bacterial density was assessed at 24 h . Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded . The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate . A significant effect on the yield was recorded at the highest aeration rate . This result was confirmed by a direct comparison in two 5-1 internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture . Possible reasons for the positive correlation between aeration rate and DJ yield are discussed.

Appl Biochem Biotechnol, 2000 May, 87(2), 117 - 25
Production of citric acid using immobilized conidia of Aspergillus niger; Bayraktar E et al.; Conidia of Aspergillus niger were immobilized in calcium alginate gel for the production of citric acid . First, the type of the preactivation medium, together with the preactivation period, was investigated . It was found that A . niger requires a 2-d preactivation period at a 0.05 g/L NH4NO3 concentration . Second, preactivated cells were used to determine the effects of nitrogen concentration and the flow rate of oxygen and air on the production of citric acid . Maximum citric acid production was attained with medium containing 0.01 g/L of NH4NO3 . The rate of citric acid production in the nitrogenous medium was 33% higher when oxygen was used instead of air during the production phase . This corresponds to an increase of 85% when compared to production when neither oxygen nor air was fed into the system . In the nonnitrogenous medium citric acid concentration remained similar regardless of the use of air or oxygen . However, in the nonnitrogenous production medium, citric acid production was not influenced considerably when oxygen was used instead of air . The advantage of using immobilized cells is that production is achieved easily in the continuous system . Therefore, citric acid production was also tested using a packed-bed bioreactor, and an increase in productivity by a factor of 22 was achieved compared to the batch system.

J Appl Microbiol, 2000 Jul, 89(1), 85 - 9
Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture; Fiedurek J et al.; A novel method for increasing dissolved oxygen concentration in culture media has been developed . It involves adding hydrogen peroxide (H2O2) to the medium which is then decomposed to oxygen and water by catalase . Some factors affecting oxygenation of culture were investigated . Maximal oxygen concentration occurred in 50 ml of the medium containing 0-2 g wet mycelium and 0.2% glucose at pH 5.0 . A new apparatus for automated addition of H2O2 to the bioreactor to keep the dissolved oxygen concentration constant over the range 1-100% +/- 2% was tested . A significant increase (over sixfold) of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable (30% +/- 2%).

Tissue Eng, 2000 Apr, 6(2), 183 - 8
Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate biopolyester for use in tissue engineering; Sodian R et al.; Previously, we reported the implantation of a single tissue engineered leaflet in the posterior position of the pulmonary valve in a lamb model . The major problems with this leaflet replacement were the scaffold's inherent stiffness, thickness, and nonpliability . We have now created a scaffold for a trileaflet heart valve using a thermoplastic polyester . In this experiment, we show the suitability of this material in the production of a biodegradable, biocompatible scaffold for tissue engineered heart valves . A heart valve scaffold was constructed from a thermoplastic elastomer . The elastomer belongs to a class of biodegradable, biocompatible polyesters known as polyhydroxyalkanoates (PHAs) and is produced by fermentation (Metabolix Inc., Cambridge, MA) . It was modified by a salt leaching technique to create a porous, three-dimensional structure, suitable for tissue engineering . The trileaflet heart valve scaffold consisted of a cylindrical stent (1 mm X 15 mm X 20 mm I.D.) containing three valve leaflets . The leaflets were formed from a single piece of PHA (0.3 mm thick), and were attached to the outside of the stent by thermal processing techniques, which required no suturing . After fabrication, the heart valve construct was allowed to crystallize (4 degrees C for 24 h), and salt particles were leached into doubly distilled water over a period of 5 days to yield pore sizes ranging from 80 to 200 microns . Ten heart valve scaffolds were fabricated and seeded with vascular cells from an ovine carotid artery . After 4 days of incubation, the constructs were examined by scanning electron microscopy . The heart valve scaffold was tested in a pulsatile flow bioreactor and it was noted that the leaflets opened and closed . Cells attached to the polymer and formed a confluent layer after incubation . One advantage of this material is the ability to mold a complete trileaflet heart valve scaffold without the need for suturing leaflets to the conduit . Second advantage is the use of only one polymer material (PHA) as opposed to hybridized polymer scaffolds . Furthermore, the mechanical properties of PHA, such as elasticity and mechanical strength, exceed those of the previously utilized material . This experiment shows that PHAs can be used to fabricate a three-dimensional, biodegradable heart valve scaffold.

Tissue Eng, 2000 Apr, 6(2), 119 - 27
A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration; Hadlock T et al.; Alternatives to autografts have long been sought for use in bridging neural gaps . Many entubulation materials have been studied, although with generally disappointing results in comparison with autografts . The purpose of this study was to design a more effective neural guidance conduit, to introduce Schwann cells into the conduit, and to determine regenerative capability through it in an in vivo model . A novel, fully biodegradable polymer conduit was designed and fabricated for use in peripheral nerve repair, which approximates the macro- and microarchitecture of native peripheral nerves . It comprised a series of longitudinally aligned channels, with diameters ranging from 60 to 550 microns . The lumenal surfaces promoted the adherence of Schwann cells, whose presence is known to play a key role in nerve regeneration . This unique channel architecture increased the surface area available for Schwann cell adherence up to five-fold over that available through a simple hollow conduit . The conduit was composed of a high-molecular-weight copolymer of lactic and glycolic acids (PLGA) (MW 130,000) in an 85:15 monomer ratio . A novel foam-processing technique, employing low-pressure injection molding, was used to create highly porous conduits (approximately 90% pore volume) with continuous longitudinal channels . Using this technique, conduits were constructed containing 1, 5, 16, 45, or more longitudinally aligned channels . Prior to cellular seeding of these conduits, the foams were prewet with 50% ethanol, flushed with physiologic saline, and coated with laminin solution (10 microg/mL) . A Schwann cell suspension was dynamically introduced into these processed foams at a concentration of 5 X 10(5) cells/mL, using a simple bioreactor flow loop . In vivo regeneration studies were carried out in which cell-laden five-channel polymer conduits (individual channel ID 500 microm, total conduit OD 2.3 mm) were implanted across a 7-mm gap in the rat sciatic nerve (n = 4), and midgraft axonal regeneration compared with autografts (n = 6) . At 6 weeks, axonal regeneration was observed in the midconduit region of all five channels in each experimental animal . The cross-sectional area comprising axons relative to the open conduit cross sectional area (mean 26.3%, SD 10 . 1%) compared favorably with autografts (mean 23.8%, SD 3.6%) . Our methodology can be used to create polymer foam conduits containing longitudinally aligned channels, to introduce Schwann cells into them, and to implant them into surgically created neural defects . These conduits provide an environment permissive to axonal regeneration . Furthermore, this polymer foam-processing method and unique channeled architecture allows the introduction of neurotrophic factors into the conduit in a controlled fashion . Deposition of different factors into distinct regions within the conduit may be possible to promote more precisely guided neural regeneration.

Tissue Eng, 2000 Feb, 6(1), 75 - 9
New pulsatile bioreactor for in vitro formation of tissue engineered heart valves; Hoerstrup SP et al.; Two potential obstacles to the creation of implantable tissue engineered heart valves are inadequate mechanical properties (ability to withstand hemodynamic stresses) and adverse host-tissue reactions due to the presence of residual nondegraded polymer scaffold . In an attempt to address these problems, we developed an in vitro cell culture system that provides physiological pressure and flow of nutrient medium to the developing valve constructs . It is anticipated that in vitro physical stress will stimulate the tissue engineered heart valve construct to develop adequate strength prior to a possible implantation . Long-term in vitro development will be realized by an isolated and thereby contamination-resistant system . Longer in vitro development will potentially enable more complete biodegradation of the polymeric scaffold during in vitro cultivation . This new dynamic bioreactor allows for adjustable pulsatile flow and varying levels of pressure . The system is compact and easily fits into a standard cell incubator, representing a highly isolated dynamic cell culture setting with maximum sterility, optimal gas supply and stable temperature conditions especially suited for long-term experiments.

Biotechnol Bioeng, 2000 Oct 5, 70(1), 32 - 40
The fluid dynamic and shear environment in the NASA/JSC rotating-wall perfused-vessel bioreactor; Begley CM et al.; The rotating-wall perfused-vessel (RWPV) bioreactor, used for both microgravity and Earth-based cell science experiments, is characterized in terms of the fluid dynamic and fluid shear stress environment . A numerical model of the flow field is developed and verified with laser Doppler velocimeter measurements . The effects of changes in operating conditions, including rotation rates and fluid perfusion rates, are investigated with the numerical model . The operating conditions typically used for ground-based experiments (equal rotation of the inner and outer cylinders) leads to flow patterns with relatively poor mass distribution characteristics . Approximately 50% of the inlet-perfused fluid bypasses the bulk of the fluid volume and flows to the perfusion exit . For operating conditions typical in microgravity, small differential rotation rates between the inner and outer cylinders lead to greatly improved flow distribution patterns and very low fluid shear stress levels over a large percentage of the fluid volume . Differences in flow patterns for the different operating conditions are explored . Large differences in the hydrodynamic environments for operating conditions typical of true microgravity and ground-based "microgravity simulations" are demonstrated.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1505 - 11
Bacteria of an anaerobic 1,2-dichloropropane-dechlorinating mixed culture are phylogenetically related to those of other anaerobic dechlorinating consortia; Schlotelburg C et al.; A 16S-rDNA-based molecular study was performed to determine the bacterial diversity of an anaerobic, 1,2-dichloropropane-dechlorinating bioreactor consortium derived from sediment of the River Saale, Germany . Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified using conserved primers . A clone library was constructed and analysed by sequencing the 16S rDNA inserts of randomly chosen clones followed by dot blot hybridization with labelled polynucleotide probes . The phylogenetic analysis revealed significant sequence similarities of several as yet uncultured bacterial species in the bioreactor to those found in other reductively dechlorinating freshwater consortia . In contrast, no close relationship was obtained with as yet uncultured bacteria found in reductively dechlorinating consortia derived from marine habitats . One rDNA clone showed >97% sequence similarity to Dehalobacter species, known for reductive dechlorination of tri- and tetrachloroethene . These results suggest that reductive dechlorination in microbial freshwater habitats depends upon a specific bacterial community structure.

Vox Sang, 2000, 78 Suppl 2, 191 - 5
Clinical use of intravenous immunoglobulins; Nydegger UE et al.; Prophylaxis and treatment with i.v . immunoglobulins must envisage preparations from normal or hyperimmunised human donors, animals (horses and rabbits) as well as monoclonal and genetically and proteomically engineered chimeric or recombinant antibodies . The latter group of antibody sources from the bioreactor source must be seen in the context of traditional antibody therapy, including passive immunization, general antibody substitution and provision of lost immune regulatory capacities such as downregulation of complement activation, attenuation of Fc receptor apparatus as well as anti-idiotypic potential . Beyond summarizing the present evidence based indications the present review is an outlook at the doorstep for future possibilities to improve precision of antibody dependent treatments and avoiding side effects which formerly compromised widespread use.

Metab Eng, 2000 Apr, 2(2), 115 - 25
Bioreactor systems in drug metabolism: synthesis of cytochrome P450-generated metabolites; Rushmore TH et al.; In this communication, we report that suspension cultures of Sf21 insect cells, co-infected with baculovirus containing the cDNA for a single cytochrome P450 and NADPH-cytochrome P450 oxidoreductase, can be employed successfully as "bioreactors" for the synthesis of milligram quantities of cytochrome P450-generated metabolite(s) . Three standard or probe substrates for the human P450s were chosen for the initial biosynthetic experiments: testosterone, diazepam, and diclofenac . Testosterone (100 microM, 2.88 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to 6beta-hydroxytestosterone (2.3 mg) and 15beta-hydroxytestosterone (0.18 mg) . Diazepam (100 microM, 2.9 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to temazepam (1.1 mg), N-demethyldiazepam (0.35 mg), and oxazepam (0.15 mg) . Diclofenac (100 microM, 3.18 mg/100 ml), added to a 100-ml CYP2C9 bioreactor, was converted to 4'-hydroxydiclofenac (2.6 mg) . Since the goal for the development of the bioreactors was to provide a platform for both the production and subsequent purification of milligram quantities of P450-generated metabolite(s), a second 100-ml CYP2C9 bioreactor was used for the large-scale production and subsequent purification of 4'-hydroxydiclofenac . After 55 h of incubation, 7.95 mg of diclofenac was converted to 4.35 mg of 4'-hydroxydiclofenac, while 3.55 mg of unchanged diclofenac remained in the bioreactor . Using a simple preparative HPLC method, approximately 2.2 mg of 4'-hydroxydiclofenac and 1.9 mg of diclofenac were recovered from this experiment (28% yield) . These results indicate clearly that suspension cultures of Sf21 insect cells coexpressing a cytochrome P450 and NADPH-cytochrome P450 oxidoreductase can be used effectively as bioreactors for the production and subsequent purification of milligram quantities of P450-derived metabolite(s).

Biotechnol Prog, 2000 Jul-Aug, 16(4), 671 - 5
Design of a molecular chaperone-assisted protein folding bioreactor; Kohler RJ et al.; Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins . Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding . In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system . The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir . This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH) . We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates.

J Appl Physiol, 2000 Aug, 89(2), 855 - 64; discussion 848
Selected contribution: PKC activation inhibits Ca(2+) signaling in tracheal epithelial cells kept in simulated microgravity; Felix JA et al.; Microgravity has been shown to alter protein kinase C (PKC) activity; therefore, we investigated whether microgravity influences mechanically stimulated Ca(2+) signaling and ATP-induced Ca(2+) oscillations, both of which are modulated by PKC . Rabbit tracheal epithelial outgrowth cultures or suspended epithelial sheets were rotated in bioreactors to simulate microgravity . Mechanical stimulation of a single cell increased the cytosolic Ca(2+) concentration in 35-55 cells of both outgrowth cultures and epithelial sheets kept at unit gravity (G) or in simulated microgravity (smicroG) . In outgrowth cultures, 12-O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKC activator, restricted Ca(2+) "waves" to about 10 cells in unit G and to significantly fewer cells in smicroG . TPA only slightly reduced the spread of Ca(2+) waves in epithelial sheets kept in smicroG but did not inhibit Ca(2+) waves of sheets kept in unit G . In both cell preparations from both conditions, TPA inhibited ATP-induced Ca(2+) oscillations; however, the effect was more pronounced in cells kept in smicroG . These results suggest that PKC activation is more robust in cells subjected to smicroG.

Transplantation, 2000 Jul 15, 70(1), 7 - 14
Engineering of human vascular aortic tissue based on a xenogeneic starter matrix; Bader A et al.; BACKGROUND: The goal for tissue engineering of vascular grafts is the replacement of a diseased vessel with a functional and stable graft . We now introduce a new concept for the tissue engineering of vessels . The idea was to humanize a previously acellularized, but structurally intact, xenogeneic vessel by repopulation with human autologous cells . To this purpose, a gentle nondenaturing and nondeterging acellularization procedure for xenogeneic aortas was developed . This structure was reseeded with pre-expanded peripheral vascular endothelial cells (EC) and myofibroblasts using specifically designed bioreactors . METHODS: Aortas from 15-30 kg female landrace pigs were acelullarized with a 0.1% trypsin solution for between 24 and 96 hr . Human vascular cells were harvested from saphenous vein biopsy specimens . Acellularized vessels were reseeded with EC and myofibroblasts . Cell viability after reseeding was assayed by fluorescence staining . Morphologic features of the acellularized matrix and tissue engineered vessel was assayed by transmission and scanning electron microscopy and histologic analysis . Nitric oxide-synthetase activity was investigated by mass spectrometric analysis of bioreactor supernatants . The in vivo immune response was tested by subcutaneous implantation of acellularized porcine aortic tissue in a rat model . RESULTS: The acellularization procedure resulted in an almost complete removal of the original resident cells, and the 3-D matrix was loosened at interfibrillar zones . However, the 3-D arrangement of the matrix fibers was grossly maintained . The 3-D matrix was covered with a fully confluent human endothelial cell layer obtained by continuous stress challenge in the bioreactor . Myofibroblasts migrated into positions formerly occupied by the xenogeneic cells . Nitric oxide synthetase activity was maintained in the bioartificial graft . T-lymphocyte and CD18 positive leukocyte infiltrate were greatly reduced after acellularization of porcine aortic specimens after implantation in the rat . CONCLUSIONS: Porcine vessels were acellularized and consecutively fully repopulated with human EC and myofibroblasts . This approach may eventually lead to the engineering of vessels immunologically acceptable to the host using a relatively short preparation period of 2-3 weeks . We expect matrix turnover in vivo leading to a gradual assimilation of the matrix structure by the host mediated by the hosts autologous cells.

Biotechnol Bioeng, 2000 Sep 20, 69(6), 597 - 606
Modeling solid-to-solid biocatalysis: integration of six consecutive steps; Michielsen MJ et al.; A quantitative model for the conversion of a solid-substrate salt to a solid-product salt in a batch bioreactor seeded with product crystals is presented . The overall process consists of six serial steps (with dissolution and crystallization each in themselves complex multistep processes): solid-salt dissolution, salt dissociation into an ionic substrate and a counter-ion, bioconversion accompanied by biocatalyst inactivation, complexation of the ionic product with the counter-ion, and salt crystal growth . In the model, the consecutive steps are integrated, including biocatalyst inactivation and assuming that salt dissociation and complexation of ions are at equilibrium . Model parameters were determined previously in separate independent experiments . To validate the model, either dissolved or solid Ca-maleate was converted to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes in a batch bioreactor seeded with Ca-D-malate crystals . The model very well predicted the concentrations of all components in the liquid phase (Ca-maleate, Ca(2+), maleate(2-), D-malate(2-), and Ca-D-malate) and the amounts of the solid phases (Ca-maleate . H(2)O and Ca-D-malate . 3H(2)O), especially when high initial amounts of Ca-maleate . H(2)O and Ca-D-malate . 3H(2)O were present .

J Biomech Eng, 2000 Jun, 122(3), 252 - 60
Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels; Mauck RL et al.; Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair . Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue . In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls . A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks . Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001) . This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa) . Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).

Ann Thorac Surg, 2000 Jul, 70(1), 140 - 4
Tissue engineering of heart valves: in vitro experiences; Sodian R et al.; BACKGROUND: Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo . Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate . In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve . METHODS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA) . The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques . The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days . Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology . RESULTS: It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor . The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure . The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold . Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions . CONCLUSIONS: Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold . The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Artif Organs, 2000 Jul, 24(7), 519 - 25
Design of a fluidized bed bioartificial liver; Legallais C et al.; The aim of this study was to design a bioreactor for extracorporeal liver supply containing alginate beads in a fluidized bed regimen . The objective was to achieve a satisfactory mixing into the bioreactor to promote the potential exchanges and mass transfers . First, we checked whether both present phases (solid: alginate beads; liquid: saline solution at 20 degrees C) might allow for this fluidization . Then the optimal design was defined as a function of the required operating conditions, bead volume, and perfusion flow rate; the bioreactor cross section and height especially needed to be adjusted . The efficient fluidization, under optimized conditions, was proven through the follow-up of the head losses generated by the fluidized bed . Criteria for scaling up were also determined.

Biorheology, 2000, 37(1-2), 141 - 7
Modulation of the mechanical properties of tissue engineered cartilage; Martin I et al.; Cartilaginous constructs have been grown in vitro using chondrocytes, biodegradable polymer scaffolds, and tissue culture bioreactors . In the present work, we studied how the composition and mechanical properties of engineered cartilage can be modulated by the conditions and duration of in vitro cultivation, using three different environments: static flasks, mixed flasks, and rotating vessels . After 4-6 weeks, static culture yielded small and fragile constructs, while turbulent flow in mixed flasks induced the formation of an outer fibrous capsule; both environments resulted in constructs with poor mechanical properties . The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels had the highest fractions of glycosaminoglycans and collagen (respectively 75% and 39% of levels measured in native cartilage), and the best mechanical properties (equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential were all about 20% of values measured in native cartilage) . Chondrocytes in cartilaginous constructs remained metabolically active and phenotypically stable over prolonged cultivation in rotating bioreactors . The wet weight fraction of glycosaminoglycans and equilibrium modulus of 7 month constructs reached or exceeded the corresponding values measured from freshly explanted native cartilage . Taken together, these findings suggest that functional equivalents of native cartilage can be engineered by optimizing the hydrodynamic conditions in tissue culture bioreactors and the duration of tissue cultivation.

Biochem, Eng . J. . 2000 Aug 1, 6(1), 65 - 74
Characterization of energy conversion based on metabolic flux analysis in mixotrophic liverwort cells, Marchantia polymorpha; Hata J et al.; In order to characterize the contributions of respiratory and photosynthetic actions to energy conversions, the mixotrophic cells of Marchantia polymorpha were cultivated in the medium containing 10kg/m(3) glucose as an organic carbon source . The cultures were conducted with the supply of ordinary air (0.03% CO(2)) at constant incident light intensities of 50 and 180W/m(2) . From the results of metabolic analysis, it was found that the cell yield based on ATP synthesis was estimated to be 6.3x10(-3)kg-dry cells/mol-ATP in these cultures . Under the examined conditions, energy conversion efficiency through respiration was larger than that through photosynthesis, and efficiency of overall energy conversion to ATP was maximized when the sum of energies from glucose and light captured by the cells was approximately 7.2x10(5)J/(hkg-dry cells) . Taking into account the efficiency of overall energy conversion, a batch culture of M . polymorpha in a bioreactor was carried out by regulating incident light intensity ranging from 9 to 58W/m(2) . In the culture with light regulation, the cell yield of 6.2x10(-9)kg-dry cells/J was achieved on the basis of energy provided to the system throughout the culture, and this value was 2.3 and 9.3 times as large as those obtained in the cultures under constant incident light intensities of 50 and 180W/m(2), respectively.

Biochem, Eng . J. . 2000 Aug 1, 6(1), 25 - 31
Single versus multiple bioreactor scale-up: economy for high-value products; Rouf SA et al.; The economy of scaling-up a bioreactor by increasing the number of units was investigated with respect to an integrated flowsheet . For the production of t-PA from animal cells, a base case flowsheet using a single large bioreactor was compared to a multiple bioreactor case . Simulation of the complete flowsheets for the two cases showed that a multiple bioreactor approach to scale-up increases the return of investment (ROI) of the base process by 122% . This enormous increase in ROI results from the smaller size of the downstream units compared to the base case, since downstream processing accounts for about 80% of the total cost for high value products like t-PA . Proper scheduling of the downstream units allowed sharing of the equipment by the bioreactors . A breakdown of the equipment purchase cost showed that cost related to cell culture equipment increased from 14% for the base case to about 37% for the multiple bioreactor case . The contribution from chromatography columns to the total equipment purchase cost, on the other hand, decreased from 52 to 33%.

Biotechniques, 2000 Jul, 29(1), 128 - 32, 134-6, 138 passim
Antibody engineering at the millennium; Gavilondo JV et al.; It has been almost 100 years since von Behring and Kitasato received the first Nobel prize for the discovery of passive immunotherapy and nearly 25 years since Kohler and Milstein first reported hybridoma technology . In the 15 years since Mullis and co-workers described PCR, a number of discoveries and technologies have converged to produce a renaissance in antibody therapeutics . Our vision of antibodies as tools for research--useful for the prevention, detection and treatment of disease--has been revolutionized by these recent advances . This review specifically focuses on what is now called antibody engineering and includes chimeric and humanized antibodies, immunoglobulin fragments, antibody libraries, antibody fusion proteins and transgenic organisms as bioreactors . As a consequence of refinements in antibody technology, the field of genetically engineered immunoglobulins has matured into an elegant and important drug and reagent development platform.

J Pharm Sci, 2000 Aug, 89(8), 979 - 90
Biomedical application of immobilized enzymes; Liang JF et al.; Reports on chemical immobilization of proteins and enzymes first appeared in the 1960s . Since then, immobilized proteins and enzymes have been widely used in the processing of variety of products and increasingly used in the field of medicine . Here, we present a review of recent developments in immobilized enzyme use in medicine . Generally speaking, the use of immobilized enzyme in medicine can be divided into two major categories: biosensors and bioreactors . A brief overview of the evolution of the biosensor and bioreactor technology, of currently existing applications of immobilized enzymes, of problems that researchers encountered, and of possible future developments will be presented .

J Gastroenterol, 2000, 35(7), 493 - 503
Developments in bioartificial liver research: concepts, performance, and applications; Nagamori S et al.; As an alternative to liver transplantation, numerous researchers have been working toward the goal of development of a fully functional artificial liver . In recent years, artificial liver support systems have been advocated as interim treatments for patients awaiting hepatocyte replacement therapy or liver transplantation; so-called "bridging" treatments . It is recognized that an effective artificial liver system requires: (1) a viable and highly functional hepatocyte cell line, (2) a suitable bioreactor environment and peripheral control systems, and (3) an effective extracorporeal circulatory system to incorporate an artificial liver system . Conventional systems have, however, suffered from various drawbacks, including incompatibility of cell cultures derived from non-human cells, insufficient cell proliferation, rapid deterioration of cellular function due to an impoverished cellular environment, and lack of system scalability . A newly established artificial liver system overcomes many of these problems and demonstrates a long-term capacity to maintain multiple liver-specific functions, such as protein synthesis, enzyme activity, and drug metabolism, both quantitatively and qualitatively . The present review provides an overview of the concepts underpinning artificial liver systems, the performance of presently available systems and the practical applications of available systems and those in development.

Arthritis Rheum, 2000 Jul, 43(7), 1580 - 90
Response of engineered cartilage tissue to biochemical agents as studied by proton magnetic resonance microscopy; Potter K et al.; OBJECTIVE: To test the hypothesis that magnetic resonance imaging (MRI) results correlate with the biochemical composition of cartilage matrix and can therefore be used to evaluate natural tissue development and the effects of biologic interventions . METHODS: Chondrocytes harvested from day-16 chick embryo sterna were inoculated into an MRI-compatible hollow-fiber bioreactor . The tissue that formed over a period of 2-4 weeks was studied biochemically, histologically, and with MRI . Besides natural development, the response of the tissue to administration of retinoic acid, interleukin-1beta (IL-1beta), and daily dosing with ascorbic acid was studied . RESULTS: Tissue wet and dry weight, glycosaminoglycan (GAG) content, and collagen content all increased with development time, while tissue hydration decreased . The administration of retinoic acid resulted in a significant reduction in tissue wet weight, proteoglycan content, and cell number and an increase in hydration as compared with controls . Daily dosing with ascorbic acid increased tissue collagen content significantly compared with controls, while the administration of IL-1beta resulted in increased proteoglycan content . The water proton longitudinal and transverse relaxation rates correlated well with GAG and collagen concentrations of the matrix as well as with tissue hydration . In contrast, the magnetization transfer value for the tissue correlated only with total collagen . Finally, the self-diffusion coefficient of water correlated with tissue hydration . CONCLUSION: Parameters derived from MR images obtained noninvasively can be used to quantitatively assess the composition of cartilage tissue generated in a bioreactor . We conclude that MRI is a promising modality for the assessment of certain biochemical properties of cartilage in a wide variety of settings.

Enzyme Microb Technol, 2000 Aug 1, 27(3-5), 205 - 211
Candida cloacae oxidation of long-chain fatty acids to dioic acids; Green KD et al.; Candida cloacae cells oxidize long-chain fatty acids to their corresponding dicarboxylic acids (dioic acids) at rates dependent on their chain length and degree of saturation . This is despite the well-known toxicity of the fatty acids . Among the saturated substrates, the oxidation is limited to lauric acid (C12) . The addition of pristane (5% v/v), which acts as an inert carrier for the poorly water-soluble substrate, boosts the oxidation of lauric acid to a rate that is comparable to that of dodecane . When dissolved in pristane, myristic (C14) and palmitic (C16) acids are effective carbon sources for C . cloacae, but dioic acid production is very low . Media glucose concentration and pH also influence cell growth and productivity . After the glucose is depleted, oxidation is optimal at a low pH . A two-phase (pristane/water) reaction was tested in a 2-l stirred tank bioreactor in which growth and oxidation were separated . A 50% w/w conversion of lauric acid (10 g/l) to dodecanedioic acid was achieved . The bioreactor also alleviated poor mass transfer characteristics experienced in shake flasks.

Biotechnol Bioeng, 2000 Sep 5, 69(5), 548 - 58
Growth of Ca-D-malate crystals in a bioreactor; Michielsen MJ et al.; To develop a bioreactor for solid-to-solid conversions, the conversion of solid Ca-maleate to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes was studied . In a bioreactor seeded with product (Ca-D-malate) crystals, growth of Ca-D-malate crystals is the last step in the solid-to-solid conversion and is described here . Crystal growth is described as a transport process followed by surface processes . In contrast to the linear rate law obeyed by the transport process, the surface processes of a crystal-growth process can also obey a parabolic or exponential rate law . Growth of Ca-D-malate crystals from a supersaturated aqueous solution was found to be surface-controlled and obeyed an exponential rate law . Based on this rate law, a kinetic model was developed which describes the decrease in supersaturation due to Ca-D-malate crystal growth as a function of the constituent ions, Ca(2+) and D-malate(2-) . The kinetic parameters depended on temperature, but, as expected (surface-controlled), they were hardly affected by the stirring speed .

Lett Appl Microbiol, 2000 Jul, 31(1), 39 - 41
Relief from glucose interference in microcin B17 biosynthesis by growth in a rotating-wall bioreactor; Fang A et al.; Glucose interference in production of microcin B17 by Escherichia coli ZK650 was decreased sevenfold by growth in a ground-based rotating-wall bioreactor operated in the simulated microgravity mode as compared with growth in flasks . When cells were grown in the bioreactor in the normal gravity mode, relief from glucose interference was even more dramatic, amounting to a decrease in glucose interference of over 100-fold.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 55 - 9
{Enzymatic resolution of racemic naproxen in a low aqueous-organic biphase system}; Xin JY et al.; A stereoselective hydrolysis of the racemic naproxen methyl ester by immobilized lipase from Candida rugosa in a low aqueous-organic biphase system was studied . Support polar, water content, the logP value of organic phase and product inhibition effected the activity of immobilized enzyme . According to these reaction conditions, a low aqueous-organic biphase system for the continuous production of (S)-(+)-Naproxen was developed . The reaction was carried out in a continuous-flow closed-loop 50 mL stirred bioreactor packed with YWG-C6H5, a poorly polar synthetic support on which the lipase had been immobilized by adsorption . The aqueous phase was permanently remained in the reactor associated with the immobilized enzyme particles; the organic phase containing substrate was pumped through this reactor and emerged with the products . The continous-flow stirred bioreactor containing 75 mg lipase was allowed to operate continuously for 60 days at 30 degrees C with a 25% loss of activity, 900 mg of (S)-(+)-Naproxen (eep 95%) were producted.

J Biomed Mater Res, 2000 Sep 15, 51(4), 642 - 9
Comparative study of seeding methods for three-dimensional polymeric scaffolds; Burg KJ et al.; Development of tissue-engineered devices may be enhanced by combining cells with porous absorbable polymeric scaffolds before implantation . The cells are seeded throughout the scaffolds and allowed to proliferate in vitro for a predetermined amount of time . The distribution of cells throughout the porous material is one critical component determining success or failure of the tissue-engineered device . This can influence both the successful integration of the device with the host tissue as well as the development of a vascularized network throughout the entire scaffold volume . This research sought to compare different seeding and proliferation methods to select an ideal method for a polyglycolide/aortic endothelial cell system . Two seeding environments, static and dynamic, and three proliferation environments, static, dynamic, and bioreactor, were analyzed, for a total of six possible methods . The six seeding and proliferation combinations were analyzed following a 1-week total culture time . It was determined that for this specific system, dynamic seeding followed by a dynamic proliferation phase is the least promising method and dynamic seeding followed by a bioreactor proliferation phase is the most promising.

Zh Mikrobiol Epidemiol Immunobiol, 1999 Nov-Dec, (6), 14 - 7
{The effect of methylated cyclodextrin on pertussis toxin accumulation in a Bordetella pertussis culture in a bioreactor}; Siundiukova RA et al.; The results obtained in the study of the influence methylated cyclodextrin (beta CD) on the growth of B . pertussis and the accumulation of pertussis toxin in the course of submerged batch cultivation in a bioreactor are presented . As demonstrated by these results, the presence of beta CD in the culture medium in a dose of 0.1 ml/l in the growth deceleration phase causes a tenfold increase in the synthesis of pertussis toxin by microbial cells in comparison with conditions characterized by the absence of beta CD . It cannot by ruled out that beta CD may act as a stressor which influences the synthesis of pertussis toxin, protector protein making it possible for the microbe to survive under new conditions.

Biotechnol Annu Rev, 2000, 5, 269 - 92
Transgenic farm animals: applications in agriculture and biomedicine; Yang X et al.; During the last decade, tremendous progress has been made in the area of transgenic farm animals . While there are many important transgenic farm animal applications in agriculture, funding has been very limited and progress has been rather slow in this area . Encouragingly, the potential applications of transgenic farm animals as bioreactors for producing human therapeutic proteins and as organ donors for transplantations in humans have attracted vast funding from the private sectors . Several transgenic animal products are already in various phases of clinical trials . Estimates are, that in the near future, the worlds demands on human pharmaceutical proteins may largely be met by transgenic farm animals . While there are still major challenges ahead in the area of xenotransplantation using transgenic animal organs, transgenic tissues or cells have demonstrated promising results as a potential tool for gene therapy . Recent development on cloning, embryonic stem cells and alternative transgenic methods may further expand the transgenic applications in both agriculture and biomedicine.

Cancer Res, 2000 Jun 15, 60(12), 3254 - 61
Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors; Scott AM et al.; The Lewis Y (Ley) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy . The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Ley-expressing cells of the MCF-7 breast carcinoma cell-line . The murine 3S193 showed high specificity for Ley in ELISA tests with synthetic Ley and Ley-containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells . We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI K chain . The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies . Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors . The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations . hu3S193 reacted specifically with Ley antigen, with similar avidity to the murine form . hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 microg/ml) . The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Ley-positive cells . Six i.v . doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120-130 mm3) with no significant effect on tumor growth . In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody . hu3S193 has promise for immunotherapy of Ley-positive tumors and is currently entering Phase I clinical trials.

Arch Otolaryngol Head Neck Surg, 2000 Jun, 126(6), 759 - 65
Growth of tissue-engineered human nasoseptal cartilage in simulated microgravity; Falsafi S et al.; OBJECTIVE: To evaluate the feasibility of in vitro fabrication of tissue-engineered cartilage from human nasoseptal chondrocytes for autologous reconstruction . DESIGN: Hyaline cartilage was reconstituted from chondrocyte-polyglycolic acid scaffolding constructs in a 3-dimensional mammalian cell culture cascade . This included monolayer cellular amplification, cell seeding in the spinner flask, and tissue growth in simulated microgravity . RESULTS: The quality of the fabricated cartilage analogue was found to depend on the initial cell density, duration of incubation, and bioreactor type . Dynamic seeding was nearly completed within the first 10 hours of inoculation regardless of the cell source (cryogenically preserved vs fresh chondrocytes) or presence of serum . A duration of incubation in excess of 4 weeks was required for complete matrix biosynthesis at low seeding densities in the spinner flasks . Seeding densities greater than 2.3x10(6) chondrocytes per scaffold were required for early hyaline cartilage formation as well as longer-time mature matrix regeneration . In addition, maintaining the structural integrity of the unreinforced scaffold, which is necessary for continued mature matrix regeneration, was achievable through postseeding tissue growth in simulated microgravity . CONCLUSION: Once combined with polyglycolic acid scaffolds in the bioreactor cascades that allow efficient seeding and quiescent tissue growth, human septal chondrocytes become a valuable source of reproducible ex vivo cartilage regeneration in the laboratory.

Biotechnol Bioeng, 2000 Aug 20, 69(4), 409 - 17
Microbial dynamics in a continuous stirred tank bioreactor exposed to an alternating sequence of organic compounds; Ferreira Jorge RM et al.; Microbial dynamics during aerobic biodegradation of an alternating mixture of organic compounds was investigated experimentally in a continuous stirred tank bioreactor (CSTB) . A mathematical model describing this system was developed and tested using the experimental results . A model microbial culture consisting of Pseudomonas sp . JS150, a monochlorobenzene (MCB) degrader, and Xanthobacter autotrophicus GJ10, a 1,2-dichloroethane (DCE) degrader, each with exclusive degradation capabilities, was used . The CSTB was inoculated with both microbial strains and exposed to an alternating sequence of the two compounds at noninhibitory concentrations . Concentrations of each microbial strain, of each organic compound, and of degradation product evolved, as well as specific microbial activities via oxygen uptake tests, were monitored . Reduction of the residual DCE discharged from the bioreactor after an MCB to DCE transition was successfully achieved by continuously feeding a low flow of a concentrated solution of both compounds .

Biotechnol Bioeng, 2000 Aug 20, 69(4), 401 - 8
A two-reservoir, hollow-fiber bioreactor for the study of mixed-population dynamics: design aspects and validation of the approach; Manjarrez ES et al.; A two-reservoir, membrane bioreactor for carrying out studies of mixed-population dynamics in batch fermentations is presented . Mixing requirements and design aspects for the validity of the approach are given and discussed . Equations describing mixing times between the reservoirs are presented and compared to the experimental results . The validity of the approach is demonstrated by the study of an amensalistic-type interaction, the protein-mediated killer phenomenon between two Saccharomyces cerevisiae strains . The validation consisted in the comparison between the results obtained in actual mixed culture and the results obtained by keeping the strains separated . A good agreement was found which demonstrates the viability of the designed bioreactor .

Biotechnol Bioeng, 2000 Aug 5, 69(3), 330 - 8
On-line control of fed-batch fermentation of dilute-acid hydrolyzates; Taherzadeh MJ et al.; Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast . In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures . A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor . The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate . Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced . The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER . However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF) . The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation . The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF . A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%) .

Biotechnol Bioeng, 2000 Aug 5, 69(3), 266 - 74
Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors; Geserick C et al.; A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells . The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor . An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein . Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest . However, no stably expressing clone was isolated in this study . Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles . The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture .

Biotechnol Bioeng, 2000 Jul 20, 69(2), 171 - 82
Cell damage of microcarrier cultures as a function of local energy dissipation created by a rapid extensional flow; Gregoriades N et al.; Microcarrier cultures of Chinese hamster ovary cells were subjected to a range of energy dissipations created by an abrupt contraction . These flow conditions can be characterized as a rapidly transient, extensional, and shear flow . Cell damage was measured using a lactate dehydrogenase assay . The laminar flow in the device was modeled using two commercial, computation fluid-dynamic codes: POLYFLOW and FLUENT . Cell damage was correlated to numerical values of energy dissipation . The magnitude of energy dissipation at which cell damage began to be detected, 10(4) ergs cm(-3) s(-1) (10(4) cm(2) s(-3)), is consistent with values of energy dissipation estimated in bioreactors operated under conditions which result in cell damage . This magnitude of energy dissipation is orders of magnitude lower than those values reported to cause damage to suspended animals cells which is also consistent with generally accepted experimental observations . Finally, an analysis and discussion of the presence and relative importance with re- spect to cell damage of shear vs . extensional flow is included .

Adv Biochem Eng Biotechnol, 2000, 67, 139 - 77
Effects of hydrodynamic and interfacial forces on plant cell suspension systems; Kieran PM et al.; Plant cells are perceived to be sensitive to the hydrodynamic environment in conventional bioreactors . Heightened sensitivity, relative to most bacterial cultures, is frequently attributed to larger plant cell sizes, extensive vacuolization and aggregation patterns . Early studies of shear sensitivity focused on cell lysis and/or loss of viability . More recently, an extensive array of sub-lethal responses has been identified . A fuller understanding of these sub-lytic effects may assist in the optimization of large-scale cultivation conditions . This paper reviews recent work on the hydrodynamic shear sensitivity of plant cell systems, under cultivation conditions and in purpose-built shearing devices . The relevance of different approaches to the characterization of the intensity of a given hydrodynamic environment is discussed . Indicators of cell response to hydrodynamic stress are evaluated . The potential significance of cellular defense mechanisms, observed in response to mechanical stimulants, is identified.

Adv Biochem Eng Biotechnol, 2000, 67, 123 - 37
Influence of stress on adherent cells; Kretzmer G; Stress is a broad term often used with animal cells . Frequently mechanical forces are meant using this term but chemical stress is also important cultivating animal cells . The chemical environment of the cell in a reactor have to be considered very carefully . The complexity of the medium requirements and the metabolic pathway cause very often growth limitations . Studying these limitations in order to find the reasons showed to be difficulty because of the complexity of the system . Nevertheless, glucose, glutamine, lactate and ammonia are found to be critical parameter as well as the osmotic pressure . The influence of mechanical forces on cell viability is of great importance when growing the cells in agitated systems . By far the greatest amount of work reported in the literature has been done on suspension cells but adherent cells also experience shear forces not only in bioreactors also in vivo . Therefore, most research has be done on endothelial cells but studies exists done on non-endothelial cells . The influence of shear forces on cell growth, morphology and productivity will be discussed as well as possibilities of making the cells more resistant.

Adv Biochem Eng Biotechnol, 2000, 67, 35 - 82
Particle stress in bioreactors; Henzler HJ; In many biological processes, e.g . the fermentation of cells and sensitive microorganisms or bioconversion with immobilised enzymes, low shear stress is of crucial importance for the optimal course of processes . Starting with the causes of particle stress, the following report discussed the hydrodynamic principles of the most frequently used model reactors and bioreactors, which are required for an approximate calculation of stress . The main part of the report describes the results of systematic investigations into the hydrodynamic stress on particles in stirred tanks, reactors with dominating boundary-layer flow, shake flasks, viscosimeters, bubble columns and gas-operated loop reactors . These results for model and biological particle systems permit fundamental conclusions on particle stress and the dimensions and selection of suitable bioreactors according to the criterion of particle stress.

Appl Microbiol Biotechnol, 2000 May, 53(5), 542 - 4
Suspended rice particles for cultivation of Monascus purpureus in a tower-type bioreactor; Wu WT et al.; Cultivation of Monascus purpureus (CCRC 31615) for the production of natural pigments was investigated . Traditionally, Monascus species were grown on rice by solid-state culture . For large-scale cultivation, solid-state cultures were associated with some problems such as contamination and scale-up . By using submerged cultures with rice particles, a stirred-tank fermentor was not suitable for submerged cultures as the impeller tended to break the particles into small pieces . A conventional bubble column was also unsuitable as its mixing capability was poor . In the present study, a modified bubble column with wire-mesh draft tubes was employed for the cultivation of M . purpureus . The proposed column had a shorter mixing time and a higher oxygen transfer rate relative to the conventional bubble column . The production of pigments using the proposed column was up to 80% higher than that achieved using the conventional bubble column.

Curr Opin Microbiol, 2000 Jun, 3(3), 244 - 7
Microbial physiological state at low growth rate in natural and engineered ecosystems; Konopka A; Microbes often grow in nature and bioreactors at very low growth rates . However, the physiological consequences at low growth rates have not been explored as completely as at faster growth rates or under starvation conditions . Nutrient flux to (and through) the cell surface and non-growth-dependent energy consumption (maintenance) are important considerations under these conditions . The biomass recycle reactor is a system to explore physiological state at low growth rate, and to optimize certain industrial process rates.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 707 - 19
Biotreatment of refinery spent-sulfidic caustic using an enrichment culture immobilized in a novel support matrix; Conner JA et al.; Sodium hydroxide solutions are used in petroleum refining to remove hydrogen sulfide (H2S) and mercaptans from various hydrocarbon streams . The resulting sulfide-laden waste stream is called spent-sulfidic caustic . An aerobic enrichment culture was previously developed using a gas mixture of H2S and methyl-mercaptan (MeSH) as the sole energy source . This culture has now been immobilized in a novel support matrix, DuPont BIO-SEP beads, and is used to bio-treat a refinery spent-sulfidic caustic containing both inorganic sulfide and mercaptans in a continuous flow, fluidized-bed column bioreactor . Complete oxidation of both inorganic and organic sulfur to sulfate was observed with no breakthrough of H2S and < 2 ppmv of MeSH produced in the bioreactor outlet gas . Excessive buildup of sulfate (> 12 g/L) in the bioreactor medium resulted in an upset condition evidenced by excessive MeSH breakthrough . Therefore, bioreactor performance was limited by the steady-state sulfate concentration . Further improvement in volumetric productivity of a bioreactor system based on this enrichment culture will be dependent on maintenance of sulfate concentrations below inhibitory levels.

Adv Drug Deliv Rev, 1998 Aug 3, 33(1-2), 15 - 30
Culture of organized cell communities; Vunjak-Novakovic G et al.; Cells cultured in vitro will tend to retain their differentiated phenotype under conditions that resemble their natural in vivo environment, for example, when cultured on polymer scaffolds in tissue culture bioreactors . In this chapter, we define organized cell communities as three-dimensional in vitro grown cell-polymer constructs that display important structural and functional features of the natural tissue . We review representative studies in which the research goal was to culture organized cell communities resembling cartilage, bone, skeletal muscle or cardiac-like tissue . These constructs can potentially serve as tissue equivalents for in vivo transplantation or as a model system for the in vitro testing of cell and tissue-level responses to molecular, mechanical or genetic manipulations.

Biotechnol Prog, 2000 May-Jun, 16(3), 471 - 9
Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads; Ghanem A et al.; Our overall objective is to develop a cell culture analogue bioreactor (CCA) that can be used together with a corresponding physiologically based pharmacokinetic model (PBPK) to evaluate molecular mechanisms of toxicity . The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal . The CCA bioreactor is a physical replica of the PBPK; where the PBPK specifies organs, the CCA bioreactor contains compartments with a corresponding cell type that mimics some of the characteristic metabolism of that organ . The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium . The CCA bioreactor and the model can be coupled to evaluate the plausibility of the molecular mechanism that is input into the model . This paper focuses on the design, development, and characterization of a CCA bioreactor to be used in naphthalene dose response studies . A CCA bioreactor prototype developed previously is improved upon by culturing the cells on microcarrier beads . Microcarrier beads with cells attached can form packed beds with cell culture medium perfusing the beds . In this study, two packed beds of cells, one with L2 cells (rat lung) and one with H4IIE cells (rat hepatoma), are linked in a physiologically relevant arrangement by a common recirculating cell culture medium . Studies of this CCA bioreactor presented here include mixing profiles, effect of reactor environment on cell viability and intracellular glutathione, naphthalene distribution profile, and initial naphthalene dosing studies . Unlike the prototype system there is no detectable response to naphthalene addition; in a companion paper we show that this discrepancy can be explained by differences in liquid residence times in the organ compartments . The perfusion reactor design is shown to have significant operating improvements over prototype designs.

Biotechnol Prog, 2000 May-Jun, 16(3), 462 - 70
Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors; Kunkel JP et al.; The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors . All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors . The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F) . Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) . The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation . There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides . The level of DO affects the glycosylation of this mAb . A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant . However, the quantitative results are not precise matches between the two bioreactors . The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO . There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints . The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems . The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.

Biotechnol Prog, 2000 May-Jun, 16(3), 425 - 34
Development and qualification of a novel virus removal filter for cell culture applications; Liu S et al.; Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses . The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor . A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media . This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV . Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions . The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product . Insulin and transferrin show high passage through the filter . The virus barrier filter can be autoclaved . The relatively high membrane permeability enables the use of a moderate filtration area.

Biotechnol Prog, 2000 May-Jun, 16(3), 391 - 401
Hairy root culture in a liquid-dispersed bioreactor: characterization of spatial heterogeneity; Williams GR et al.; A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots . The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7 . Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall . Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow . With root growth, a pattern of spatial heterogeneity developed in the vessel . Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor . Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor . Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed . Roots near the bottom of the vessel trapped up to 3-4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems . Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed . Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance . The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance . Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems.

Biotechnol Prog, 2000 May-Jun, 16(3), 378 - 84
Toluene removal in an automated cyclical bioreactor; Brown WA et al.; A control scheme was developed for the automation of toluene removal in a cyclical bioreactor . Toluene was added to the self-cycling fermentor by diffusion across a silicone membrane . Transient dissolved oxygen, carbon dioxide evolution, and oxidation-reduction potential (ORP) were screened as potential control variables . Through experimentation, ORP was deemed most effective . Control algorithms based on real-time estimates of the first and second derivatives of the ORP signal were tested . Although both approaches resulted in stable operation of the reactor, average toluene removal efficiencies of 95% were realized when control was based on the second derivative . This was significantly higher than the 77% efficiencies obtained when the control scheme centered on the first derivative of the transient ORP signal . The system developed was self-regulating, ensuring that a high toluene removal rate, on the order of 1.1 g h(-1), was maintained from cycle to cycle.

Biotechnol Prog, 2000 May-Jun, 16(3), 334 - 45
Combining cell culture analogue reactor designs and PBPK models to probe mechanisms of naphthalene toxicity; Ghanem A et al.; An alternative method of evaluating the toxicology of a chemical is to use cultured mammalian cells in a novel cell culture analogue reactor (CCA) together with a corresponding physiologically based pharmacokinetic model (PBPK) . The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal . The bioreactor is a physical replica of the PBPK; where the PBPK specifies an organ or tissue compartment, the bioreactor contains compartments with a corresponding cell type . The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium . The bioreactor and the model are coupled to evaluate the plausibility of the molecular mechanism that is input into the model . This concept is tested with naphthalene as a model of PAH (polycyclic aromatic hydrocarbons) toxicants . Two physically different CCA reactors were tested with naphthalene, and different results were observed . In the prototype system using cells attached to glass dilution bottles, naphthalene dosing resulted in generation of a circulating metabolite from the "liver" compartment (based on H4IIE cells from a rat hepatoma) that caused cell death in the "lung" compartment (L2 cells from a rat lung), as well as depletion of glutathione in the L2 cells . An improved CCA using packed bed reactors of microcarrier cultured cells did not show differences between naphthalene-dosed and nondosed controls . To explain the different responses of the two CCA designs, PBPKs of the two reactors were tested with variations in physical and kinetic parameters, and toxic mechanism . When the toxic metabolite of naphthalene was naphthoquinone rather than naphthalene epoxide as initially assumed, the PBPK results were consistent with the results of the two CCA designs . This result indicates that the mechanism of naphthalene toxicity in the CCAs may be mediated through naphthoquinone formation . The CCA-PBPK concept is demonstrated to be applicable to the study of toxic mechanisms . In particular, use of this approach suggests that in vitro naphthalene toxicity is mediated through the naphthoquinone metabolite.

Growth Factors, 2000, 18(1), 11 - 29
Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor; Domagala T et al.; The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy . These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC . The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR . Analytical ultracentrifugation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon et al . (1997) . Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF:sEGFR interaction and that more complex interaction mechanisms are operative . Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity . Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2-20 nM and the other with a KD of 400-550 nM.

Ultrasonics, 2000 Mar, 38(1-8), 711 - 6
Improvement of biological activity by low energy ultrasound assisted bioreactors; Schlafer O et al.; Bioreactors are broadly applied in biotechnology and wastewater treatments . The so-called advanced bioreactor systems should be optimised in a more compact, more efficient and more effective form . One method of optimisation is the improvement of the solid-liquid interface of the sludge flocs and the mass transfer rate of gas and nutrients in the liquid . This could be obtained by the intake of ultrasound in the bioreactor at a frequency of 25 kHz and a power input of 0.3 Wl-1 . An increase in the biological activity of the process investigated with alternating ultrasound as well as a decrease without ultrasound were measured.

Biochem, Eng . J. . 2000 Jul 1, 5(3), 225 - 229
Local overall volumetric gas-liquid mass transfer coefficients in gas-liquid-solid reversed flow jet loop bioreactor with a non-Newtonian fluid; Jianping W et al.; The local overall volumetric gas-liquid mass transfer coefficients at the specified point in a gas-liquid-solid three-phase reversed flow jet loop bioreactor (JLB) with a non-Newtonian fluid was experimentally investigated by a transient gassing-in method . The effects of liquid jet flow rate, gas jet flow rate, particle density, particle diameter, solids loading, nozzle diameter and CMC concentration on the local overall volumetric gas-liquid mass transfer coefficient (K(L)a) profiles were discussed . It was observed that local overall K(L)a profiles in the three-phase reversed flow JLB with non-Newtonian fluid increased with the increase of gas jet flow rate, liquid jet flow rate, particle density and particle diameter, but decreased with the increase of the nozzle diameter and CMC concentration . The presence of solids at a low concentration increased the local overall K(L)a profiles, and the optimum of solids loading for a maximum profile of the local overall K(L)a was found to be 0.18x10(-3)m(3) corresponding to a solids volume fraction, varepsilon(S)=2.8%.

Curr Microbiol, 2000 Jun, 40(6), 356 - 61
Diversity of cyanobacterial hydrogenases, a molecular approach; Tamagnini P et al.; In an effort to elucidate the diversity of cyanobacterial hydrogenases, we used a molecular approach . Filamentous strains from a broad range of sources were screened for the presence of hup (uptake hydrogenase), xisC (rearrangement within hupL), and hox (bidirectional hydrogenase) genes . As expected, an uptake hydrogenase seems to be present in all N(2)-fixing cyanobacteria . On the other hand, no evidence was found for the presence of a conventional bidirectional enzyme in several strains . Similarly, the presence of xisC is not a characteristic shared by all the heterocyst-forming cyanobacteria . Although tempting, it is not possible to establish a correlation between the presence/absence of the bidirectional hydrogenase and the occurrence of xisC . The natural molecular variation of hydrogenases in cyanobacteria is certainly a field to explore, both to understand the physiological functions of the respective enzymes and to identify a genetic background to be used when constructing a strain for photobiological H(2) production in a bioreactor.

Biotechnol Bioeng, 2000 Jul 5, 69(1), 74 - 82
Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process; Yang JD et al.; Controlled feeding of nutrient supplements to a cell culture to enhance monoclonal antibody productivity has been practiced widely in high-yield, fed-batch processes . In this study, a similar feeding concept has been applied to a perfused culture and evaluated for the effects on bioreactor productivity and product quality . Our experimental results show that, by using such a "controlled-fed perfusion" approach, the volumetric antibody productivity (antibody per liter per day) was significantly increased by nearly twofold over the perfusion process, and surpassed fed-batch and batch processes by almost tenfold . The substantial boost in the overall productivity is attributable primarily to the combined effects of increased cell density as well as reduced product dilution . Both were achieved through careful nutrient supplementation in conjunction with metabolite minimization . As the manufacturing process evolved from roller bottles to the controlled-fed perfusion bioreactor system, the immunoreactivity and the cDNA sequences of the antibody were well preserved . However, the product glycosylation distribution patterns did alter . The controlled-feed perfusion process demonstrated a unique encompassment of the advantages of fed-batch and perfusion methods; that is, high product concentration with high volume throughput . Therefore, it may be very suitable for large-scale production of monoclonal antibodies .

Biotechnol Bioeng, 2000 Jul 5, 69(1), 39 - 46
Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field; Castelletti L et al.; A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field . With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale . Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism . The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion . To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent . The weight average molecular size in these conditions has been found to be ca . 200,000 Da . These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time . As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time . This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism .

J Mammary Gland Biol Neoplasia, 1998 Jul, 3(3), 337 - 50
The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins; Clark AJ; A variety of transgenic animal species are being used to produce recombinant proteins . The general approach is to target the expression of the desired protein to the mammary gland using regulatory elements derived from a milk protein gene and then collect and purify the product from milk . Promoter sequences from a number of different milk protein genes have been used to target expression to the mammary gland, although significant problems remain with regard to achieving transgene expression levels consistent with commercial exploitation . The mammary gland appears to be capable of carrying out the complex posttranslational modifications . such as glycosylation and gamma-carboxylation required for the biological activity and stability of specific proteins . Effective purification protocols have been established and products produced by this route have now entered clinical trials.

J Chromatogr A, 2000 Apr 7, 874(2), 207 - 15
Determination of anionic surfactants during wastewater recycling process by ion pair chromatography with suppressed conductivity detection; Levine LH et al.; A direct approach utilizing ion pairing reversed-phase chromatography coupled with suppressed conductivity detection was developed to monitor biodegradation of anionic surfactants during wastewater recycling through hydroponic plant growth systems and fixed-film bioreactors . Samples of hydroponic nutrient solution and bioreactor effluent with high concentrations (up to 120 mS electrical conductance) of inorganic ions can be analyzed without pretreatment or interference . The presence of non-ionic surfactants did not significantly affect the analysis . Dynamic linear ranges for tested surfactants {Igepon TC-42, ammonium lauryl sulfate, sodium laureth sulfate and sodium alkyl (C10-C16) ether sulfate} were 2 to approximately 500, 1 to approximately 500, 2.5 to approximately 550 and 3.0 to approximately 630 microg/ml, respectively.

J Biotechnol, 2000 Apr 14, 79(1), 27 - 37
Influence of controlled glucose oscillations on a fed-batch process of recombinant Escherichia coli; Lin HY et al.; The influence of glucose oscillations on cell growth and product formation of a recombinant Escherichia coli culture producing a heterologous alpha-glucosidase was studied in fed-batch cultures in a laboratory bioreactor . Glucose oscillations were created by an on/off-feeding mode in either fast cycles (1 min) or slow cycles (4 min) and compared to a process with constant glucose addition . The study indicates that glucose oscillations influence the product stability and the overgrowth of plasmid-free cells if such cultures are not performed under continuous pressure for selection of plasmid-containing cells . Although the glucose uptake capacity decreased after induction of the recombinant alpha-glucosidase in all cultures performed, the up-growth of plasmid-free cells during the production phase was strongly inhibited by fast oscillations . In contrast, plasmid-free cells grew up when constant feeding or slow cycles were applied . Our data suggest that the various feed protocols effect the specific carbon dioxide formation rate differently, with the highest production of carbon dioxide in the cultivations with fast cycles . In connection to product formation the initial alpha-glucosidase accumulation was the same in all cultures, but the stability of the product was significantly lower in the cultivation with slow cycles . Our results from laboratory experiments are discussed in relation to the mixing situation in large-scale bioreactors.

J Biotechnol, 2000 Apr 14, 79(1), 1 - 11
A novel perfusion system for animal cell cultures by two step sequential sedimentation; Wen ZY et al.; A novel perfusion system was developed for high density culture of animal cells . The system consists of an airlift bioreactor, a setting tank and a flat settler . Both the settling tank and flat settler have two connecting pipes for transporting the cells from and back to the reactor, respectively . Thus, the cell flow in the settlers can be controlled in uni-direction, avoiding the countercurrent flow of the cells . During perfusion cultures, the cells firstly settled in the settling tank, then, unsettled cells in the tank were transferred to the flat settler for re-settling . With the application of the system to hybridoma cell cultures, it was found that the maximum viable cell density, monoclonal antibody concentration and average productivity were 1.31 x 107 cells ml-1, 400 mg l-1 and 461 mg l-1 d-1, respectively, which were much higher than those of a batch culture . Both theoretical analysis and experimental results showed a much higher separation efficiency in such a two-step sedimentation system than that in a conventional one-step sedimentation system . In addition, the volumetric ratio of the sedimentation devices to the culture volume in our developed system is much lower, which may be potentially useful on an industrial-scale.

Biochem, Eng . J. . 2000 Jun 1, 5(2), 157 - 164
Light intensity distribution in the externally illuminated cylindrical photo-bioreactor and its application to hydrogen production by Rhodobacter capsulatus; Katsuda T et al.; The light distribution in the externally illuminated cylindrical photo-bioreactor for production of hydrogen by a photosynthetic bacterium Rhodobacter capsulatus ST-410 was estimated . The estimation was performed on the basis of the Matsuura and Smith's diffuse model {1} . In the diffuse model, the incident light rays are assumed to proceed in every direction and the local intensity is calculated as the sum of the intensities of light . Since Lambert-Beer's law, extensively used in photometry, was not useful for explaining the decrease in the intensity of light by the biomass, an empirical expression was used . The measurement of the intensities from every direction was conducted in an externally illuminated cylindrical photo-bioreactor having an inner diameter of 60mm and a working volume of 550ml . The obtained results confirmed our estimation . The light distribution was applied to estimate the hydrogen production by R . capsulatus ST-410 using the same photo-bioreactor . The overall hydrogen-production rate was successfully estimated.

Biochem, Eng . J. . 2000 Jun 1, 5(2), 101 - 107
Optimizing initial plasmid copy number distribution for improved protein activity in a recombinant fermentation; Patnaik PR; Recombinant bacterial cells in a fermentation broth rarely contain the same number of plasmids, even though this simplification is often used . Recent work has however indicated limitations of the simplified approach . Based on these studies, the distribution of plasmid copy numbers per cell has been represented macroscopically here in a Gaussian form for the fraction of biomass as a function of the copy number . Applying this distribution and an experimentally validated kinetic model to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) synthesis by Escherichia coli containing the plasmid pBR Eco gap, it is seen that GAPDH production in a batch fermentation is maximized by a particular initial (non-zero) copy number variance and an optimal duration . To implement this distribution in a bioreactor, it is suggested that the profile may be discretized, inocula corresponding to the mean copy number of each fraction prepared, and then combined to obtain the seed culture.

Biochem, Eng . J. . 2000 Jun 1, 5(2), 89 - 100
Studies on the modeling and simulation of a sequential bienzymatic reaction system immobilized in emulsion liquid membrane; Pal P et al.; Experiments have been carried out to study the reaction engineering behavior of the liquid membrane-encapsulated, sequential bienzymatic reaction system, n 2n glucose . A dynamic mathematical model, free from adjustable parameters, has been developed taking into account peri-emulsion mass transfer, intra-emulsion diffusion, membrane-related mass transfer limitations and substrate and product inhibitions . A finite difference-based, user-friendly software has been developed to solve the model equations . Experimental data satisfactorily correlate with the model . While it is understood that study of sequential bienzymatic reaction system immobilized in emulsion liquid is essential for their industrial exploitation, reaction engineering behavior of such a system in presence of both substrate and product inhibitions has not yet been reported in the literature . Therefore, the model predictions of the present investigations are expected to pave the way for scale-up and design of industrial bioreactors in this field.

J Biotechnol, 2000 Apr 28, 79(2), 127 - 36
Production of antifungal recombinant peptides in Escherichia coli; Gavit P et al.; Antifungal peptides derived from the human bactericidal/permeability-increasing protein (BPI) were produced in Escherichia coli as fusion proteins with human BoneD . Bacterial cultures transformed with the gene encoding the fusion protein were grown to a high cell density (OD(600)>100), and induced with L-arabinose to initiate product expression . Fusion protein accumulated into cytoplasmic inclusion bodies and recombinant peptide was released from BoneD by acid hydrolysis at an engineered aspartyl-prolyl dipeptide linker . Acid hydrolysis of purified inclusion bodies at pH <2.6 followed Arrhenius kinetics and did not require prior inclusion body solubilization in detergents or denaturants . Surprisingly, at pH <2.6 and 85 degrees C, cell lysis and aspartyl-prolyl hydrolysis with concomitant peptide release occurred simultaneously . Bacterial cultures were, therefore, adjusted to approximately pH 2.6 with HCl directly in the bioreactor and incubated at elevated temperature . Peptide, which is soluble in the aqueous acidic environment, was separated from the insoluble material and purified using column separation techniques . Recombinant peptide was separated from the hydrolyzed bioreactor culture with >76% recovery and a final peptide purity of >97% . Antifungal peptide prepared by recombinant and solid phase synthesis methods demonstrated similar activity against Candida sp . in a broth microdilution assay.

Appl Microbiol Biotechnol, 2000 Apr, 53(4), 383 - 9
Axenic cultivation of anoxygenic phototrophic bacteria, cyanobacteria, and microalgae in a new closed tubular glass photobioreactor; Hai T et al.; A low-cost closed tubular glass photobioreactor allowing axenic cultivation of phototrophic microorganisms was constructed . Standard glass tubes were arranged in a helical array providing a working volume of 80 1 . The glass tubes were connected with a degassing chamber, which also provided ports for measuring and regulating oxygen supply, pH, foam, and optical density and for adding substrates and antifoam agents as well as disposing of vent gas . A pump module allowed agitation of the medium in the bioreactor at a laminar flow rate of 1.5 m/s . Upstream of the pump module a gas inlet was located, allowing efficient mixing of the used gases with the medium . The temperature of the medium was controlled by a Pt-100 sensor and by a heat exchanger with an effective surface of 0.12 m2 connected to an external thermostat . Irradiation was provided by three light panels each consisting of ten fluorescent tubes . The entire photobioreactor - apart from the light panels and motor - could be sterilized at 121 degrees C in an autoclave . In addition to a detailed description of this photobioreactor, we report on first experiments to cultivate the anoxygenic phototrophic bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum, the oxygenic phototrophic cyanobacterium Synechocystis sp . strain PCC6803, and the microalga Chlorella sp . in this photobioreactor.

Biotechnol Bioeng, 2000 Jun 20, 68(6), 697 - 704
Observations on the shear damage to different animal cells in a concentric cylinder viscometer; Mardikar SH et al.; A clear distinction is made between damage to the population of cells and damage to individual cells on exposure to shear stress . Work on mechanical damage to animal cells in suspension is reported for six different cell lines . Precisely controlled shears of 1 Pa, 10 Pa, and 100 Pa were generated in a viscometer and distortions in morphology of the cells-for instance, the formation of transient pores, cytoplasmic extrusions, and ghost-cell membranes-are presented with photographic evidence . Low shears are shown to be just as damaging as the higher shears, although the type of damage is different . It follows that bioreactors should be operated at intermediate shear levels for optimal yield . A mechanism to account for the unexpected stability of animal cells at intermediate levels of shear is presented .

Biotechnol Bioeng, 2000 Jun 20, 68(6), 660 - 4
Hydrolysis of glucosinolates using nylon-immobilized myrosinase to produce pure bioactive molecules; Leoni O et al.; Bioactive compounds were produced from natural glucosinolates, secondary plant metabolites, using myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) isolated from ripe seeds of Sinapis alba . The enzyme was immobilized on granular nylon 6.6 with the crosslinking technique . Immobilized myrosinase displayed extraordinary operational and storage stability . Using a small thermostatted continuous packed-bed bioreactor, the enzyme activity was unchanged after 15 days of continuous use at 37 degrees C and after >1 year of storage at room temperature . The bioreactor was particularly efficient in producing pure isothiocyanates, but it was less efficient for pure nitrile production .

Biotechnol Bioeng, 2000 Jun 20, 68(6), 589 - 93
Power consumption in shaking flasks on rotary shaking machines: I . Power consumption measurement in unbaffled flasks at low liquid viscosity; Buchs J et al.; In this first article of a series a new method is introduced that enables the accurate determination of the power consumption in a shaking flask . The method is based on torque measurements in the drive and appropriate compensation of the friction losses . The results for unbaffled shaking flasks at low viscosities are presented after varying shaking frequency, flask size, filling volume, shaking diameter, and surface quality (hydrophilic and hydrophobic) of the inner flask walls . The order of magnitude of the values of power consumption in shaking flasks is equal to, or even higher than, the values typical for agitated tank bioreactors . A physically based model equation for shaking flasks is derived that introduces a modified power number and a resulting constant as the only fitting parameter . With this equation, the measured results are correlated with sufficient accuracy . For the first time, comprehensive data for the power consumption in unbaffled shaking flasks at low viscosity is available, giving a detailed picture of the influences of the different variables .

Biotechnol Bioeng, 2000 Jun 5, 68(5), 584 - 7
Contamination of a high-cell-density continuous bioreactor; Domingues L et al.; Continuous fermentations were carried out with a recombinant flocculent Saccharomyces cerevisiae strain in an airlift bioreactor . Once operating under steady state at a dilution rate of 0.45 h(-1), the bioreactor was contaminated with Escherichia coli cells . The faster growing E . coli strain was washed out of the bioreactor and the recombinant, slower growing flocculating S . cerevisiae strain remained as the only species detected in the bioreactor . Flocculation, besides allowing for the realization of high-cell-density systems with corresponding unusual high productivity, may be used as a selective property for controlling some contamination problems associated with prolonged continuous operation .

Biotechnol Bioeng, 2000 Jun 5, 68(5), 548 - 56
A hollow-fiber membrane bioreactor for the removal of trichloroethylene from the vapor phase; Pressman JG et al.; A hollow-fiber membrane bioreactor was used to separate trichloroethylene (TCE) from a gaseous waste stream with subsequent cometabolic biodegradation by a pure culture of Methylosinus trichosporium OB3b PP358 . The two-stage bioreactor system was successfully operated for 20 days . PP358 was grown in a continuous-flow chemostat and circulated through the fiber lumen of a hollow-fiber membrane module (HFMM), while TCE contaminated air (141 to 191 microg/L) was pumped through the HFMM shell . Between 54% -84% TCE transfer and 92%-96% TCE cometabolism were obtained in the HFMM reactor loop . Short shell-residence times, 1.6 to 5.0 minutes, demonstrated quick throughput of TCE contaminated air . Best-fit computer modeling of the biological experiments estimated mass transfer coefficients between 2.0 x 10(-3) cm/min and 5.6 x 10(-3) cm/min . The average pseudo-first-order biodegradation rate constant for the biological experiments was 0.46 L/mg TSS/d . These results demonstrate that the hollow-fiber membrane bioreactor represents an attractive technology for the bioremediation of gaseous waste streams .

Biotechnol Bioeng, 2000 Jun 5, 68(5), 473 - 87
ATR-FTIR sensor development for continuous on-line monitoring of chlorinated aliphatic hydrocarbons in a fixed-bed bioreactor; Acha V et al.; This article describes the continuous on-line monitoring of a dechlorination process by a novel attenuated total reflection-Fourier transform infrared (ATR-FTIR) sensor . This optical sensor was developed to measure noninvasively part-per-million (ppm) concentrations of trichloroethylene (TCE), tetrachloroethylene (PCE), and carbon tetrachloride (CT) in the aqueous effluent of a fixed-bed dechlorinating bioreactor, without any prior sample preparation . The sensor was based on an ATR internal reflection element (IRE) coated with an extracting hydrophobic polymer, which prevented water molecules from interacting with the infrared (IR) radiation . The selective diffusion of chlorinated compound molecules from aqueous solution into the polymer made possible their detection by the IR beam . With the exclusion of water the detection limits were lowered, and measurements in the low ppm level became possible . The best extracting polymer was polyisobutylene (PIB) in the form of a 5.8-microm thick film, which afforded a detection limit of 2, 3, and 2 . 5 mg/L (ppm) for TCE, PCE, and CT, respectively . Values of the enrichment factors between the polymer coating and the water matrix of these chloro-organics were determined experimentally and were compared individually with predictions obtained from the slopes of absorbance/concentration curves for the three analytes . Before coupling the ATR-FTIR sensor to the dechlorinating bioreactor, preliminary spectra of the chlorinated compounds were acquired on a laboratory scale configuration in stop-flow and flow-through closed-loop modes . In this way, it was possible to study the behavior and direct response of the optical sensor to any arbitrary concentration change of the analytes . Subsequently, the bioreactor was monitored with the infrared sensor coupled permanently to it . The sensor tracked the progression of the analytes' spectra over time without perturbing the dechlorinating process . To calibrate the ATR-FTIR sensor, a total of 13 standard mixtures of TCE, PCE and CT at concentrations ranging from 0 to 60 ppm were selected according to a closed symmetrical experimental design derived from a 3(2) full-factorial design . The above range of concentrations chosen for calibration reflected typical values during normal bioreactor operation . Several partial least squares (PLS) calibration models were generated to resolve overlapping absorption bands . The standard error of prediction (SEP) ranged between 0.6 and 1 ppm, with a relative standard error of prediction (RSEP) between 3 and 6% for the three analytes . The accuracy of this ATR-FTIR sensor was checked against gas chromatography (GC) measurements of the chlorocompounds in the bioreactor effluents . The results demonstrate the efficiency of this new sensor for routine continuous on-line monitoring of the dechlorinating bioreactor . This strategy is promising for bioprocess control and optimization .

J Appl Microbiol, 2000 Apr, 88(4), 655 - 62
Optimization of high-molecular-weight polycyclic aromatic hydrocarbons' degradation in a two-liquid-phase bioreactor; Marcoux J et al.; A microbial consortium degrading the high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) pyrene, chrysene, benzo{a}pyrene and perylene in a two-liquid-phase reactor was studied . The highest PAH-degrading activity was observed with silicone oil as the water-immiscible phase; 2,2,4,4,6,8, 8-heptamethylnonane, paraffin oil, hexadecane and corn oil were much less, or not efficient in improving PAH degradation by the consortium . Addition of surfactants (Triton X-100, Witconol SN70, Brij 35 and rhamnolipids) or Inipol EAP22 did not promote PAH biodegradation . Rhamnolipids had an inhibitory effect . Addition of salicylate, benzoate, 1-hydroxy-2-naphtoic acid or catechol did not increase the PAH-degrading activity of the consortium, but the addition of low-molecular-weight (LMW) PAHs such as naphthalene and phenanthrene did . In these conditions, the degradation rates were 27 mg l-1 d-1 for pyrene, 8.9 mg l-1 d-1 for chrysene, 1.8 mg l-1 d-1 for benzo{a}pyrene and 0.37 mg l-1 d-1 for perylene . Micro-organisms from the interface were slightly more effective in degrading PAHs than those from the aqueous phase.

Yeast, 2000 Apr, 16(6), 483 - 97
Dynamic in vivo (31)P nuclear magnetic resonance study of Saccharomyces cerevisiae in glucose-limited chemostat culture during the aerobic-anaerobic shift; Gonzalez B et al.; The purpose of this work was to analyse in vivo the influence of sudden oxygen depletion on Saccharomyces cerevisiae, grown in glucose-limited chemostat culture, using a recently developed cyclone reactor coupled with (31)P NMR spectroscopy . Before, during and after the transition, intracellular and extracellular phosphorylated metabolites as well as the pHs in the different cellular compartments were monitored with a time resolution of 2.5 min . The employed integrated NMR bioreactor system allowed the defined glucose-limited continuous cultivation of yeast at a density of 75 g DW/l and a p(O(2)) of 30% air saturation . A purely oxidative metabolism was maintained at all times . In vivo (31)P NMR spectra obtained were of excellent quality and even allowed the detection of phosphoenolpyruvate (PEP) . During the switch from aerobic to anaerobic conditions, a rapid, significant decrease of intracellular ATP and PEP levels was observed and the cytoplasmic pH decreased from 7.5 to 6.8 . This change, which was accompanied by a transient influx of extracellular inorganic phosphate (P(i)), appeared to correlate linearly with the decrease of the ATP concentration, suggesting that the cause of the partial collapse of the plasma membrane pH gradient was a reduced availability of ATP . The complete phosphorous balance established from our measurement data showed that polyphosphate was not the source of the increased intracellular P(i) . The derived intracellular P(i), ATP and ADP concentration data confirmed that the glycolytic flux at the level of glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and enolase enzymes is mainly controlled by thermodynamic constraints .

J Biotechnol, 2000 Jan 7, 76(1), 61 - 71
Monitoring cellular state transitions in a production-scale CHO-cell process using an electronic nose; Bachinger T et al.; An electronic nose is used to monitor the bioreactor off-gas composition in perfused cultivations of a CHO-cell line producing recombinant human blood coagulation factor VIII . The applicability of the electronic nose for monitoring cellular state transitions and process control is explained . It is shown that the instrument can reveal characteristic process states related to product and lactate formation, and detect microbial infections in a very early stage of the infection . The visualization of ideal process conditions is realized by using principal component analysis (PCA) and the on-line applicability of this method is outlined . The results illustrate the potential of the electronic nose as on-line sensor for ensuring product and process quality in production-scale bioprocesses.

FEMS Microbiol Lett, 2000 May 1, 186(1), 127 - 32
Physiology and continuous culture of the hyperthermophilic deep-sea vent archaeon Pyrococcus abyssi ST549; Godfroy A et al.; The deep-sea vent archaeon Pyrococcus abyssi strain ST549 was grown in batch cultures in closed bottles and by continuous culture in a gas-lift bioreactor, both in the presence and in the absence of elemental sulfur . Growth on carbohydrates, proteinaceous substrates and amino acids was investigated . The disaccharides maltose and cellobiose were shown not to be able to enhance growth suggesting that P . abyssi ST549 is unable to use them as carbon sources . By contrast, proteinaceous substrates such as peptone and brain heart infusion were shown to be very good substrates for the growth of P . abyssi ST549 and allowed growth at high steady-state cell densities in continuous culture . Growth on brain heart infusion was shown to require additional nutrients when sulfur was not present in the culture medium . Growth on amino acids only took place in the presence of sulfur . These results indicate that sulfur plays an important role in the metabolism and energetics of P . abyssi ST549.

Biochim Biophys Acta, 2000 May 1, 1474(3), 273 - 82
A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins; Bragonzi A et al.; Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins . Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA . Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins . Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes . Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line . A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e . interferon-gamma and the tissue inhibitor of metalloproteinases-1 . Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.

Crit Rev Biotechnol, 2000, 20(1), 1 - 15
Penicillin fermentation: mechanisms and models for industrial-scale bioreactors; Patnaik PR; Even after many years of research and industrial practice, the production of penicillin G in fed-batch fermentation by Penicillium crysogenum continues to attract research interest . There are many reasons: the commercial and therapeutic importance of penicillin and its derivatives, the complexity of cell growth, and the impact of engineering variables, the last of which are significant in large bioreactors but are not yet fully understood . Extensive research has generated new information on the mechanisms of cellular reactions and morphological features of the mycelia and their role in the synthesis of the product . Given a choice of mechanisms, models of different degrees of complexity, for both cellular differentiation and bioreactor performance, have been proposed . The more complex models require and provide more information . They are also more difficult to evaluate and apply in automatic control systems for production-scale bioreactors . The present review considers the evolution of recent knowledge and models from this perspective.

Biochem J, 2000 May 1, 347 Pt 3, 771 - 9
Mutational analysis of the N-linked glycosylation sites of the human insulin receptor; Elleman TC et al.; Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform . The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function . The effects of these mutations on proreceptor processing into alpha and beta subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells . The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined . The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin . The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation . Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (Delta6) and 16+295+337+418+730+743+881 (Delta7a), seemed normal . The third construct, 16+337+418+514+730+743+881 (Delta7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of Delta7b that was correctly processed showing insulin-stimulated autophosphorylation . The mutations of Delta6 and Delta7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain . The Delta6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

Biotechnol Prog, 2000 Mar-Apr, 16(2), 302 - 4
Continuous ethanol production from concentrated wood hydrolysates in an internal membrane-filtration bioreactor; Lee WG et al.; Continuous culture for the production of ethanol from wood hydrolysate was carried out in an internal membrane-filtration bioreactor . The hydrolysate medium was sterilized at a relatively low temperature of 60 degrees C with the intention of reducing the formation of inhibitory compounds during the sterilization . The maximum ethanol concentration and productivity obtained in this study were 76.9 g/L and 16.9 g/L-h, respectively, which were much higher than those (57.2-67 g/L and 0.3-1.0 g/L-h) obtained in batch cultures using hydrolysate media sterilized at 60 degrees C . The productivity was also found to be much higher than that (6.7 g/L-h) obtained in a continuous cell retention culture using a wood hydrolysate sterilized at 121 degrees C . These results show that the internal membrane-filtration bioreactor in combination with low-temperature sterilization could be very effective for ethanol production from wood hydrolysate.

Biotechnol Prog, 2000 Mar-Apr, 16(2), 189 - 98
NADH-Regulated metabolic model for growth of Methylosinus trichosporiumOB3b . Cometabolic degradation of trichloroethene and optimization of bioreactor system performance; Sipkema EM et al.; A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system . Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels . The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion . It is based on the metabolic pathways, includes Michaelis-Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor . Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane . The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells . In growth-based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash-out of suspended cultures at low TCE loads (below microM inlet concentrations) . Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash-out . Pulsed addition of TCE (0.014-0.048 mM) leads to a complete loss of viability . However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33-0.64 mM) . This emphasizes the inefficient use of enzyme activity in growth-based systems . A comparison of growth-based and similar non-growth-based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter . Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two-step system can be eliminated . This results in complete utilization of enzyme activity and thus in a very effective treatment system.

Biotechnol Bioeng, 2000 May 20, 68(4), 381 - 8
Enhanced growth of Sf-9 cells to a maximum density of 5.2 x 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture; Elias CB et al.; Significant improvement in cell growth and protein production has been achieved in Sf-9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bedard et al., 1994; Chan et al., 1998) . The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner . Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 x 10(7) cells/mL of Sf-9 cells in a 3.5 L bioreactor . Production of recombinant protein beta-galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 x 10(6)cells/mL . Specific productivity could be maintained at cell densities as high as 14 x 10(6) cells/mL . The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added . On-line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities .

Biotechnol Bioeng, 2000 May 5, 68(3), 328 - 38
Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor; Carswell KS et al.; Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells . The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems . Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells . Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation . Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates . For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined . No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures . The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks . This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest .

Int J Artif Organs, 2000 Feb, 23(2), 104 - 10
Comparative analysis of metabolism of medium- and plasma perfused primary pig hepatocytes cultured around a 3-D membrane network; Unger JK et al.; Culture media are frequently used in the evaluation of metabolical functions of hepatocytes in hybrid liver support systems (hLSS) . However, media compositions differ substantially from those of plasma . Therefore, our study was designed to investigate whether current in vitro studies with medium are suitable to assess the metabolical competence of hLSS-cultures during clinical application as well as to explore whether the cell nutrition with medium provides a suitable modus operandi for stand by cultivation . Paired bioreactor cultures were perfused with either Williams' Medium E (MPB) or human plasma (PPB) . About 6x108 primary pig hepatocytes (>97% viability) were cultured in three laboratory scale bioreactors designed according to Gerlach's bioreactor-concept . Different perfusion protocols were initiated after a standardised period allowing for cell attachment and reorganisation in aggregates . Whereas patterns of enzyme release were similar in both protocols the metabolical behaviour was different between MPB (anabolic state) and PPB (catabolic state) . Furthermore, compared to MPB the lidocaine-MEGX-tests for PPB demonstrated lower MEGX-concentrations and a different reaction pattern . We conclude that the nutrition of hepatocytes with medium during the stand by period itself might influence the cell function and subsequently the efficacy of the hLSS-treatment during clinical application.

Bioseparation, 1999, 8(1-5), 201 - 7
Direct coupling of expanded bed adsorption with a downstream purification step; Beck JT et al.; In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA) . EBA combines clarification, purification, and concentration of the product into a single step . The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent . This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography . Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment . The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column . Scale-down was simple and could be automated . Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost.

J Biotechnol, 2000 Mar 10, 78(2), 123 - 37
Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles; Janssen M et al.; The light regime inside a photobioreactor is characterized by a light gradient with full (sun)light at the light-exposed surface and darkness in the interior of the bioreactor . Consequently, depending on the mixing characteristics, algae will be exposed to certain light/dark cycles . In this study the green alga Chlamydomonas reinhardtii was cultivated under five different light regimes: (1) continuous illumination; (2) a square-wave light/dark cycle with a light fraction (epsilon) of 0.5 and a duration (t(c)) of 6.1 s; (3) epsilon=0.5, t(c)=14.5 s; (4) epsilon=0.5, t(c)=24.3 s and (5) epsilon=0.8, t(c)=15.2 s . The biomass yield on light energy, protein per photons, decreased under light/dark cycles (epsilon=0 . 5) in comparison to continuous light (CL), from 0.207 (CL) to 0.117-0.153 g mol(-1) (epsilon=0.5) . Concomitantly, the maximal specific photosynthetic activity, oxygen production per protein, decreased from 0.94 (CL) to 0.64-0.66 g g(-1) h(-1) (epsilon=0.5) . Also the quantum yield of photochemistry, yield of the conversion of light energy into chemical energy, decreased from 0.47 (CL) to 0 . 23 (epsilon=0.5, t(c)=24.3 s) . Apparently, C . reinhardtii is not able to maintain a high photosynthetic capacity under medium-duration light/dark cycles and since specific light absorption did not change, light utilization efficiency decreased in comparison to continuous illumination.

Biosens Bioelectron, 2000 Feb, 14(12), 895 - 905
Fiber optic monitoring of carbamate pesticides using porous glass with covalently bound chlorophenol red; Xavier MP et al.; An optical fiber biosensor for the determination of the pesticides propoxur (Baygon) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described . A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes . The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG-immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle . In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution . The performance of the biosensor has been optimized using a flow injection system . The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l(-1) and from 0.03 to 0.50 mg l(-1), respectively . The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng) . Reproducibility, stability and interference studies of the optical device are reported . The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed.

J Nucl Med, 2000 Mar, 41(3), 556 - 64
A cell-culture reactor for the on-line evaluation of radiopharmaceuticals: evaluation of the lumped constant of FDG in human glioma cells; Noll T et al.; A fluidized-bed cell-culture reactor with on-line radioactivity detection was developed for the in vitro evaluation of radiopharmaceuticals . The technique was applied to measure the dependency of the lumped constant (LC) of FDG on the glucose concentration in the culture medium in a human glioma cell line . METHODS: Human glioblastoma cells (86HG39) immobilized in open porous microcarriers were cultivated in a continuously operating fluidized-bed bioreactor . At different glucose concentrations in the culture medium, step inputs (0.1 MBq/mL) of FDG were performed and the cellular uptake of FDG was measured on-line and compared with analyzed samples . From these results, the LC of FDG and its dependency on the glucose concentration were calculated . RESULTS: This fluidized-bed technique enabled precise and reproducible adjustment of all relevant experimental parameters, including radiotracer time-concentration course, medium composition, pH, dissolved oxygen and temperature under steady-state conditions, and an on-line determination of the intracellular radiotracer uptake . The immobilized glioma cells formed stable, 3-dimensional, tumor-like spheroids and were continuously proliferating, as proven by an S-phase portion of 25%-40% . For further examination of the cells, an enzymatic method for detachment from the carriers without cellular destruction was introduced . In the FDG experiments, a significant dependency of the LC on the glucose level was found . For normoglycemic glucose concentrations, the LC was determined to be in the range of 0.7+/-0.1, whereas in hypoglycemia LC increased progressively up to a value of 1.22+/-0.01 at a glucose concentration of 3 mmol/L . CONCLUSION: The bioreactor represents an improved in vitro model for the on-line evaluation of radiotracers and combines a wide range of experimental setups and 3-dimensional, tissue-like cell cultivation with a technique for on-line radioactivity detection.

Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 446 - 450
Enzymatic esterification of ethanol by an immobilised Rhizomucor miehei lipase in a perforated rotating disc bioreactor; Oliveira AC et al.; A perforated rotating disc bioreactor was developed to perform the esterification of ethanol with oleic acid, catalyzed by a lipase from Rhizomucor miehei immobilized by adsorption on to a hydrophobic support-Accurel EP700 . The bioreactor with total recirculation operated at an optimum agitation rate of 400 rev./min . The experimental results, in this condition, were predict by a kinetic model using the constants obtained in the batch (Erlenmeyer flasks) assays: a catalytic constant, k(cat) = 5.78 mmol/h . mg protein; a Michaelis constant for ethanol, K(m(Et)) = 1.20 M; a Michaelis constant for oleic acid, K(m(Ol)) = 1.16 x 10(-8) M, and a dissociation constant of the ethanol-lipase complex, K((Et)) = 9.46 x 10(7) M . The efficiency of conversion gradually decreased during continuous operation of the reactor . The enzymatic activity decayed according to a first order deactivation model and the integrated equations of a continuous stirred tank reactor (CSTR) and a plug flow reactor (PFR) . A half-life time of the lipase of about 10 days and a deactivation constant of 0.003 h(-1) were obtained in the present system.

Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 402 - 405
Investigation of acid proteinase biosynthesis by the fungus Humicola lutea 120-5 in an airlift bioreactor; Aleksievaa P et al.; Acid proteinase production using filamentous fungus Humicola lutea 120-5 was studied under batch and continuous fermentation conditions in an airlift bioreactor . A comparison with proteinase production by fungal cells, cultivated in stirred tank bioreactor was made . The process performance in both fermentation devices was similar with respect to substrate utilization, biomass, and enzyme concentration . Continuous acid proteinase production was achieved for 14 days at an optimal dilution rate of 0.05/h with maximum specific activity of 90 U/mg DW of mycelia and yield of 38 U/mg glucose . The volumetric productivity (50 U/ml . h) was approximately 3 times higher than this of the batch system . All continuous experiments were carried out without any bacterial contamination, due to the low pH (3.0-3.5) during the process . The "pellet" type growth of the fungus in the airlift reactor prevented the system from plugging with filaments.

Biotechnol Bioeng, 2000 Apr 20, 68(2), 153 - 9
Bioartificial kidney . II . A convective flow model of a hollow fiber bioartificial renal tubule; Moussy Y; An existing model for volume transport in the rat proximal tubule was modified and applied to a bioartificial renal tubule . The predicted volume transport agrees well with experimental data . The volume transport model was coupled to the analytic solutions of flow in the bioartificial renal tubule bioreactor, operated in the open-shell mode with perfusion in both the lumen and surrounding shell . The results suggest that the performance of a multifiber bioreactor can be improved by controlling shell inlet conditions and fiber spacing . These results have important implications for the design and analysis not only for the bioartificial renal tubule bioreactor but also for the general case of hollow fiber bioreactors operated in the open-shell mode with perfusion in both the lumen and surrounding shell .

Biotechnol Bioeng, 2000 Apr 20, 68(2), 142 - 52
Bioartificial kidney . I . Theoretical analysis of convective flow in hollow fiber modules: application to a bioartificial hemofilter; Moussy Y; Analytical expressions describing convective flow in a continuous arteriovenous hollow fiber hemofilter were developed . In the lumen of the hollow fiber membrane, existing analytical expressions were applied to describe velocity profiles and pressure . For flow in the shell (the extracapillary space separating the fibers), analytical expressions for the radial and axial velocity profiles and pressure distribution were derived by first finding the stream function . The expressions are based on a similarity solution . Previous analyses of ultrafiltration have either ignored osmotic pressure or assumed constant shell pressure . In this paper, the axial variation in lumen pressure, shell pressure, and osmotic pressure were accounted for . The predicted filtration rates agree well with the experimental results . This flow model is general enough to describe flow in hollow fiber membrane systems employed as bioreactors (e.g., for cell cultures and as bioartificial organs) and as separators (e.g., ultrafiltration and microfiltration) operating in the open-shell mode . The results were applied to determine the design of an optimally functioning bioartificial hemofilter for use ex vivo or in vivo .

Biotechnol Bioeng, 2000 Apr 20, 68(2), 127 - 35
Mathematical modeling as a tool to investigate the design and operation of the zymotis packed-bed bioreactor for solid-state fermentation; Mitchell DA et al.; Zymotis bioreactors for solid-state fermentation (SSF) are packed-bed bioreactors with internal cooling plates . This design has potential to overcome the problem of heat removal, which is one of the main challenges in SSF . In ordinary packed-bed bioreactors, which lack internal plates, large axial temperature gradients arise, leading to poor microbial growth in the end of the bed near the air outlet . The Zymotis design is suitable for SSF processes in which the substrate bed must be maintained static, but little is known about how to design and operate Zymotis bioreactors . We use a two-dimensional heat transfer model, describing the growth of Aspergillus niger on a starchy substrate, to provide guidelines for the optimum design and operation of Zymotis bioreactors . As for ordinary packed-beds, the superficial velocity of the process air is a key variable . However, the Zymotis design introduces other important variables, namely, the spacing between the internal cooling plates and the temperature of the cooling water . High productivities can be achieved at large scale, but only if small spacings between the cooling plates are used, and if the cooling water temperature is varied during the fermentation in response to bed temperatures .

Aust N Z J Surg, 2000 Feb, 70(2), 127 - 31
A biodialysis system for liver support tested in a porcine hepatic failure model; Sheil AG et al.; BACKGROUND: A practical liver support system for patients in fulminant hepatic failure (FHF) remains a needed therapeutic modality . A new method of bioartificial liver support, the liver biodialysis system (LBDS), is described . METHODS: Porcine hepatocytes, removed from direct contact with the treated subject's circulation, are in culture in a bioreactor which is combined in a dialysis circuit for patient treatment . The LBDS was tested in a porcine ischaemic hepatic failure model . RESULTS: The viable hepatocyte content of the bioreactor was 2.49 +/- 0.72 x 10(10) . Cells remained viable in culture throughout the experiments (30 +/- 3 h) without evidence of immunological damage . A decrease in the degree of accumulation in the blood of ammonia (P < 0.02) and of 14 amino acids (P < 0.001) was achieved by the LBDS . Cerebral perfusion pressure was maintained at significantly higher levels in LBDS-treated animals (P < 0.05) . CONCLUSIONS: In the LBDS, hepatocytes in large numbers and satisfactory culture conditions in a bioreactor have sustained viability and function . When combined in a dialysis circuit for the treatment of FHF pigs, immune reactions between the blood and hepatocytes were prevented and beneficial metabolic effects were observed.

Blood, 2000 Mar 15, 95(6), 2169 - 74
Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer; Stiff P et al.; The collection of small aliquots of bone marrow (BM), followed by ex vivo expansion for autologous transplantation may be less morbid, and more cost-effective, than typical BM or blood stem cell harvesting . Passive elimination of contaminating tumor cells during expansion could reduce reinoculation risks . Nineteen breast cancer patients underwent autotransplants exclusively using ex vivo expanded small aliquot BM cells (900-1200 x 10(6)) . BM was expanded in media containing recombinant flt3 ligand, erythropoietin, and PIXY321, using stromal-based perfusion bioreactors for 12 days, and infused after high-dose chemotherapy . Correlations between cell dose and engraftment times were determined, and immunocytochemical tumor cell assays were performed before and after expansion . The median volume of BM expanded was 36.7 mL (range 15.8-87.0) . Engraftment of neutrophils greater than 500/microL and platelets greater than 20,000/microL were 16 (13-24) and 24 (19-45) days, respectively; 1 patient had delayed platelet engraftment, even after infusion of back-up BM . Hematopoiesis is maintained at 24 months, despite posttransplant radiotherapy in 18 of the 19 patients . Transplanted CD34(+)/Lin(-) (lineage negative) cell dose correlated with neutrophil and platelet engraftment, with patients receiving greater than 2.0 x 10(5) CD34(+)/Lin(-) cells per kilogram, engrafting by day 28 . Tumor cells were observed in 1 of the 19 patients before expansion, and in none of the 19 patients after expansion . It is feasible to perform autotransplants solely with BM cells grown ex vivo in perfusion bioreactors from a small aliquot . Engraftment times are similar to those of a typical 1000 to 1500 mL BM autotransplant . If verified, this procedure could reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which small stem cell doses are available, eg, cord blood transplants . (Blood . 2000;95:2169-2174)

Int J Pharm, 2000 Mar 20, 197(1-2), 233 - 8
Gene expression in an intact ex-vivo skin tissue model following percutaneous delivery of cationic liposome-plasmid DNA complexes; Birchall JC et al.; The skin represents an attractive site for the localised gene therapy of dermatological pathologies and as a potential antigen bioreactor following transdermal delivery . Potential also exists for the gene therapy of skin as a cosmetic intervention . The most exploited non-viral gene delivery system involves the complexation of cationic liposomes with plasmid DNA (pDNA) to form lipid:pDNA vectors that protect the DNA from nuclease-mediated degradation and improve transgene-cell interactions . Despite numerous studies examining the potential for these vectors in delivering genes to a variety of keratinocyte models, investigations into the topical application of such complexes to intact skin tissue is limited . This ex-vivo study, conducted with intact skin tissue derived from hairless mice, provides quantitative confirmation that topical administration of cationic lipid:pDNA complexes can mediate uptake and expression of reporter pDNA (33-fold higher compared with control) in viable epidermal tissue . The ex-vivo study design provides for intact skin tissue that has not been subjected to depilatory procedures of potential detriment to stratum corneum barrier function, and can be utilised for the quantitative and efficient examination of a potentially wide range of non-viral gene vectors designed for epidermal expression.

Biotechnol Bioeng, 2000 Apr 5, 68(1), 59 - 70
Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers; Werner A et al.; Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS . The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively . For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L . The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L . After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L . With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L . The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively . During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L . At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped . Concerning their metabolic activity the cells predominantly consumed glutamine and glucose . During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor . Maximal oxygen consumption was 1950 nmol/(10(6) cells . day) . HepZ cells resisted a 4-day long chilling period at 9.5 degrees C . The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL . Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency . Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system .

Biotechnol Bioeng, 2000 Mar 20, 67(6), 775 - 90
Technological problems in cultivation of plant cells at high density . Reprinted from Biotechnology and Bioengineering, Vol . XXII, No . 6, Pages 1203-1218 (1981)
Tanaka H.
Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated . Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded . The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems . Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec . The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion) . The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M) . The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.

Appl Environ Microbiol, 2000 Mar, 66(3), 1190 - 4
Propachlor removal by Pseudomonas strain GCH1 in an immobilized-cell system; Martin M et al.; A bacterial strain capable of growing on propachlor (2-chloro-N-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques . The strain isolated, designated GCH1, was classified as a member of the genus Pseudomonas . Washed-cell suspensions of strain GCH1 accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol . Pseudomonas strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 +/- 0.15 micromol h(-1) . Gene expression did not require induction but was subject to catabolite expression . Acetanilide was a growth substrate with a yield of 0.56 +/- 0.02 mg of protein micromol(-1) . GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system . Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor . The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.

FEMS Microbiol Lett, 2000 Mar 1, 184(1), 113 - 8
Aerobic nitrate respiration in a nitrite-oxidising bioreactor; McDevitt C et al.; The ability of heterotrophic bacteria in a nitrite-oxidising bioreactor to respire with nitrate as an electron acceptor was examined . Approximately 70% of 1000 heterotrophic isolates were able to express a nitrate reductase . A detailed survey of 15 isolates showed that five expressed the azide-insensitive nitrate reductase encoded by the napA gene . A two-round PCR amplification of the napA gene using degenerate PCR primers and DNA sequence analysis of these products confirmed the presence of this gene in the positive isolates . Partial 16S rDNA products and napA products were amplified from the biomass in the bioreactor and denaturing gradient gel electrophoresis of these products identified 21 distinct ribotypes and 12 distinct napA sequences . The results show that the ability to respire with nitrate as an electron acceptor under aerobic conditions is widespread among the heterotrophic population of this bioreactor.

J Biotechnol, 2000 Feb 17, 77(2-3), 275 - 86
Hydrolysis of oleuropein by recombinant beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus immobilised on chitosan matrix; Briante R et al.; The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C) . Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor . The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction . Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate . The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1) . For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach . In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h) . The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.

J Cell Biochem, 2000 Feb, 77(1), 127 - 34
Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions; Lewis ML et al.; Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized . The objective of this ground-based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions . We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP-70 . Applying the reverse transcriptase-polymerase chain reaction (RT-PCR), we probed for HSP-70 message in human acute T-cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42 degrees C) . Temperature elevation, as the positive control, significantly increased HSP-70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression . We found a fourfold increase in heat shock message in serum-starved cells . Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments . We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP-70 gene expression . In addition, we found RT-PCR useful for evaluating stress in cultured cells .

J Chromatogr A, 2000 Jan 14, 866(2), 173 - 81
Synthesis of a silica-bonded bovine serum albumin s-triazine chiral stationary phase for high-performance liquid chromatographic resolution of enantiomers; Zhang Q et al.; A novel method of synthesizing protein chiral stationary phase (protein-CSP) is proposed with 2,4,6-trichloro-1,3,5-triazine as the activator . The bovine serum albumin (BSA) based chiral columns (150 x 4.6 mm I.D.) were prepared successfully within 8 h . With tryptophan as the probe solute, it was observed that the BSA immobilized by this method had a better ability to distinguish enantiomers than that activated by glutaric dialdehyde . This may be due to the well-maintained BSA conformation and the larger amount of BSA immobilized on the silica gel . The BSA-CSP prepared by this method was relatively stable under experimental conditions, and the resolution of 13 chiral compounds was achieved . The coupling reaction in this method is mild, reliable and reproducible; it is also suitable for the immobilization of various biopolymers in the preparation of bioreactor, biosensor and affinity chromatography columns.

ASAIO J, 2000 Jan-Feb, 46(1), 107 - 10
Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valves; Sodian R et al.; A crucial factor in tissue engineering of heart valves is the type of scaffold material . In the following study, we tested three different biodegradable scaffold materials, polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), and poly-4-hydroxybutyrate (P4HB), as scaffolds for tissue engineering of heart valves . We modified PHA and P4HB by a salt leaching technique to create a porous matrix . We constructed trileaflet heart valve scaffolds from each polymer and tested them in a pulsatile flow bioreactor . In addition, we evaluated the cell attachment to our polymers by creating four tubes of each material (length equals 4 cm; inner diameter, 0.5 cm), seeding each sample with 8,000,000 ovine vascular cells, and incubating the cell-polymer construct for 8 days (37 degrees C and 5% CO2) . The seeded vascular constructs were exposed to continuous flow for 1 hour . Analysis of samples included DNA assay before and after flow exposure, 4-hydroxyproline assay, and environmental scanning electron microscopy (ESEM) . We fabricated trileaflet heart valve scaffolds from porous PHA and porous P4HB, which opened and closed synchronously in a pulsatile bioreactor . It was not possible to create a functional trileaflet heart valve scaffold from PGA . After seeding and incubating the PGA-, PHA-, and P4HB-tubes, there were significantly (p < 0.001) more cells on PGA compared with PHA and P4HB . There were no significant differences among the materials after flow exposure, but there was a significantly higher collagen content (p < 0.017) on the PGA samples compared with P4HB and PHA . Cell attachment and collagen content was significantly higher on PGA samples compared with PHA and P4HB . However, PHA and P4HB also demonstrate a considerable amount of cell attachment and collagen development and share the major advantage that both materials are thermoplastic, making it possible to mold them into the shape of a functional scaffold for tissue engineering of heart valves.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2097 - 101
The expression of proUK in Escherichia coli: the vgb promoter replaces IPTG and coexpression of argU compensates for rare codons in a hypoxic induction model; Jiang L et al.; The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector . As the proUK gene contains 2% AGG/AGA codons, which is much higher than the normal frequency in E . coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments . In E . coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK . To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter--which is known to be activated by the onset of hypoxic conditions--controls the T7RNA polymerase gene expression . Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies . Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.

Biotechnol Prog, 2000 Jan-Feb, 16(1), 102 - 8
A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: cell viability and tissue-specific functions; De Bartolo L et al.; When designing an extracorporeal hybrid liver support device, special attention should be paid to providing the architectural basis for reconstructing a proper cellular microenvironment that ensures highest and prolonged functional activity of the liver cells . The common goal is to achieve high cell density culture and to design the bioreactor for full-scale primary liver cell cultures under adequate mass transfer conditions . An important aim of this study was to evaluate the biochemical performance of a flat membrane bioreactor that permits high-density hepatocyte culture and simultaneously to culture cells under sufficient oxygenation availability conditions comparable to the in vivo-like microenvironment . In such a bioreactor pig liver cells were cultured within an extracellular matrix between oxygen-permeable flat-sheet membranes . In this investigation we used a novel scaled-up prototype consisting of up to 20 modules in a parallel mode . Each module was seeded with 2 x 10(8) cells . Microscopic examination of the hepatocytes revealed morphological characteristics as found in vivo . Cell concentration increased in the first days of culture, as indicated by DNA measurements . The performance of the bioreactor was monitored for 18 days in terms of albumin synthesis, urea synthesis, ammonia elimination, and diazepam metabolism . The ability of the hepatocytes to synthesize albumin and urea increased during the first days of culture . Higher rates of albumin synthesis were obtained at day 9 and remained at a value of 1.41 pg/h/cell until day 18 of culture . The rate of urea synthesis increased from 23 ng/h/cell to 28 ng/h/cell and then remained constant . Cells eliminated ammonia at a rate of about 56 pg/h/cell, which was constant over the experimental period . Hepatocytes in the bioreactor metabolized diazepam and generated three different metabolites: nordiazepam, temazepam, and oxazepam . The production of such metabolites was sustained until 18 days of culture . These results demonstrated that the scale-up of the bioreactor was assessed, and it could be demonstrated that the device design aimed at the reconstruction of the liver-specific tissue architecture supported the expression of liver-specific functions of primary pig liver cells.

Adv Space Biol Med, 1999, 7, 163 - 212
Electrophoresis in space; Bauer J et al.; Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed . Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground . Some studies have shown that resolution is also increased . However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation . Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity . If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found . Several free fluid electrophoresis devices are now available . Most have been tried at least once in microgravity . Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing . The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity . Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit . Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware . A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers . The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface.

J Biotechnol, 2000 Jan 21, 76(2-3), 197 - 205
Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing; Levy MS et al.; Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes . Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA . Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA . This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor . Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose . In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate . Contamination levels dropped from over 27% to below 1% as measured by Southern analysis . Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.

J Biotechnol, 2000 Jan 21, 76(2-3), 147 - 55
Optimization of gibberellic acid production by immobilized Gibberella fujikuroi mycelium in fluidized bioreactors; Escamilla EM et al.; An orthogonal experimental design L9 (3(4)) was used to investigate effects of temperature, pH, C:N ratio (glucose-C, NH4Cl-N) and concentrations of rice flour on production of gibberellic acid by Gibberella fujikuroi in 3.5 l fluidized bioreactors . The gibberellic acid production in a fluidized bioreactor could reach 3.90 g l(-1), more than 3-times greater than previously reported for submerged and solid fermentations . pH, rice flour concentration and C:N ratio were the factors that most influenced the production of gibberellic acid; pH being the most important . The response surface of gibberellic acid production to changes in pH and C:N ratio or rice flour concentration indicated that greatest production was found with a C:N ratio of 36.8 and pH 5 while the optimum concentration for rice flour was 2 g l(-1) and production increased with increased pH . The effect of temperature on the production of gibberellic acid was also significant and greatest production was at 30 degrees C.

Biologicals, 1999 Sep, 27(3), 203 - 15
Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing; Schenerman MA et al.; Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development . Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent . The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure . Studies were also performed to support cell line stability assessment and cell culture process validation . Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product . In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range . Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture . No other changes in microheterogeneity were apparent for the other culture conditions studied . In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product .

Biotechnol Bioeng, 2000 Mar 5, 67(5), 585 - 97
A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies; Sauer PW et al.; A fed-batch cell culture process was developed that has general applicability to all evaluated Sp2/0 (n = 8) and NS0 (n = 1) antibody-producing cell lines . The two key elements of this generic process were a protein-free concentrated feed medium, and a robust, metabolically responsive feeding strategy based on the off-line measurement of glucose . The fed-batch process was shown to perform equivalently at the 15 L development scale and 750 L manufacturing scale . Compared to batch cultures, the fed-batch process yielded a 4 . 3 fold increase in the average integral of viable cell concentration and a 1.7 fold increase in average specific antibody production rate, equivalent to a 7.6 fold increase in average final antibody concentration . The highest producing cell line reached a peak viable cell concentration of 1.0 x 10(7) cell mL(-1) and a final antibody concentration of 750 mg L(-1) in a 10 day process . For all lines evaluated, reducing bioreactor pH set point from 7.2 to 7.0 resulted in an additional 2.4 fold increase in average final antibody concentration . The optimized fed-batch process consistently yielded a volumetric productivity exceeding 50 mg L(-1) day(-1) . This generic, high-yielding fed-batch process significantly decreased development time, and increased manufacturing efficiency, thereby facilitating the clinical evaluation of numerous recombinant antibodies .

Biotechnol Bioeng, 2000 Mar 5, 67(5), 555 - 64
Part II . Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults; Mastrangelo AJ et al.; A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts . Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise . Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults . Extended periods of glucose deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells, typically rendering cultures completely nonviable . The overexpression of bcl-2 and bcl-x(L), however, was able to abrogate the cell death in BHK cultures, though to varying degrees . For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal . In contrast, bcl-x(L) overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study . CHO cultures, on the other hand, displayed similar trends in survival in response to both glucose and serum deprivation . During these studies, Bcl-x(L) was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and viable numbers . Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-x(L) . However, even during such harsh treatment, Bcl-x(L) was able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five days of exposure . Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed . Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities .

J Agric Food Chem, 2000 Jan, 48(1), 3 - 10
Synthesis of structured triacylglycerols by lipase-catalyzed acidolysis in a packed bed bioreactor; Xu X et al.; Structured triacylglycerols (ST) from canola oil were produced by enzymatic acidolysis in a packed bed bioreactor . A commercially immobilized 1,3-specific lipase, Lipozyme IM, from Rhizomucormiehei, was the biocatalyst and caprylic acid the acyl donor . Parameters such as substrate flow rate, substrate molar ratio, reaction temperature, and substrate water content were examined . High-performance liquid chromatography was used to monitor the reaction and product yields . The study showed that all of the parameters had effects on the yields of the expected di-incorporated (dicaprylic) ST products . Flow rates below 1 mL/min led to reaction equilibrium, and lower flow rates did not raise the incorporation of caprylic acid and the product yield . Incorporation of caprylic acid and the targeted di-incorporated ST was increased by approximately 20% with temperature increase from 40 to 70 degrees C . Increasing the substrate molar ratio from 1:1 to 7:1 increased the incorporation of caprylic acid and the product yield slightly . Water content in the substrate also had a mild influence on the reaction . Water content at 0.08% added to the substrate gave the lowest incorporation and product yield . The use of solvent in the medium was also studied, and results demonstrated that it did not increase the reaction rate at 55 degrees C when 33% hexane (v/v) was added . The main fatty acids at the sn-2 position of the ST were C(18:1), 54 . 7 mol %; C(18:2), 30.7 mol %; and C(18:3), 11.0 mol %.

Biotechnol Bioeng, 2000 Feb 20, 67(4), 435 - 50
Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates; Mercille S et al.; One of the key parameters in perfusion culture is the rate of medium replacement (D) . Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X(v)) . The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (Y(Xv/D)) and specific MAb productivity (q(MAb)) remain constant at higher D . To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operated within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems . In the Celligen/vortex-flow filter system, significant reductions in Y(Xv/D) and q(MAb) resulting from the use of gas sparging were observed at D > 1.57 vvd (X(v) > 15 x 10(6) cells/mL) . Through glucose supplementation, we have shown that the decrease in Y(Xv/D) encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation . Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (q(glc)) and lactate production rates (q(lac)), while no variations were observed in glutamine-consumption rates . As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions . Although a reduction in q(MAb) was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from shear stress-induced increases in q(lac), q(glc), and Y(lac/glc) . Deleterious effects of sparging on Y(Xv/D) and q(MAb) encountered in the Celligen/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 x 10(6) viable cells/mL . A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed .

Biotechnol Bioeng, 2000 Feb 5, 67(3), 274 - 82
Approach to designing rotating drum bioreactors for solid-state fermentation on the basis of dimensionless design factors; Hardin MT et al.; The development of large-scale solid-state fermentation (SSF) processes is hampered by the lack of simple tools for the design of SSF bioreactors . The use of semifundamental mathematical models to design and operate SSF bioreactors can be complex . In this work, dimensionless design factors are used to predict the effects of scale and of operational variables on the performance of rotating drum bioreactors . The dimensionless design factor (DDF) is a ratio of the rate of heat generation to the rate of heat removal at the time of peak heat production . It can be used to predict maximum temperatures reached within the substrate bed for given operational variables . Alternatively, given the maximum temperature that can be tolerated during the fermentation, it can be used to explore the combinations of operating variables that prevent that temperature from being exceeded . Comparison of the predictions of the DDF approach with literature data for operation of rotating drums suggests that the DDF is a useful tool . The DDF approach was used to explore the consequences of three scale-up strategies on the required air flow rates and maximum temperatures achieved in the substrate bed as the bioreactor size was increased on the basis of geometric similarity . The first of these strategies was to maintain the superficial flow rate of the process air through the drum constant . The second was to maintain the ratio of volumes of air per volume of bioreactor constant . The third strategy was to adjust the air flow rate with increase in scale in such a manner as to maintain constant the maximum temperature attained in the substrate bed during the fermentation .

Tissue Eng, 1999 Dec, 5(6), 573 - 82
Effects of microgravity on growing cultured skin constructs; Doolin EJ et al.; Understanding the cellular, chemical, and physical responses of cells to stimuli is critical to successfully engineering tissue . The effect of culturing a living skin equivalent (LSE) in a submerged microgravity environment was investigated . LSEs were developed by culturing normal human epidermal keratinocyte (NHEK) on a submerged fibroblast and type 1 collagen gel matrix . Once formed, LSEs were brought up to the air/liquid interface, and after 4 days, the cultures were maintained in either (a) a normal air/liquid interface (S), (b) resubmerged in media (R), (c) folded on themselves to enclose the keratinized layer (F/R), or (d) cut into 2-4-mm fragments and suspended in a state of microgravity in a NASA-designed bioreactor (B) . All groups were cultured for an average of 3 additional days . LSEs were processed for histologic evaluation . Skin cells were stained for cytokeratin to evaluate function . Images were digitally captured and processed for analysis . Parameters, including epithelial thickness, cellular areas, nuclear number, nuclear area, cytoplasmic area, and stained cytokeratin areas were measured . Removing the air/media interface significantly increased the number of NHEKs present in the skin; microgravity greatly enhanced this effect (p < 0.0001) . No significant difference in cellular function as measured by protein expression {stained cytokeratin area (micro(2)) per cell} was found among the groups, though the ratio of nuclear area was significantly increased in all three groups as compared to the S group (p = 0.00227) . In the case of the R and F/R groups, this appears due to the loss of the NHEK layer associated with those groups . Additionally, significant nuclear hypertrophy was demonstrated in the B group (p < 0.0001), and cellular hyperplasia was measured in all submerged groups as compared to static (p < 0.0001) . Elimination of the air/liquid interface enhanced proliferation of keratinocytes . This effect was further enhanced in the presence of microgravity . No significant effect on cell function was noted with the use of this microgravity environment . We hypothesize that the increased epidermal contact plays a role in this proliferation . Microgravity is also associated with nuclear and cellular hypertrophy over and above that of the submersion methods.

Int J Artif Organs, 1999 Nov, 22(11), 778 - 85
Morphological and functional evaluation of isolated rat hepatocytes in three dimensional culture systems; Puviani AC et al.; Various three-dimensional configurations, such as polyester tissue and woven-nonwoven, hydrophilic polyester fabric, either collagen-coated or uncoated, were investigated as potential scaffold for hepatocyte culture, in view of their use in bioreactors for hybrid liver support systems . Attachment, morphology and ultrastructure of primary adult rat hepatocytes were evaluated, as well as urea production and ammonium detoxification during a 24h incubation period in serum-free tissue culture medium . As control, hepatocytes were also plated onto collagen-coated dextran microcarriers and on plastic petri dishes, either collagen-coated or uncoated . In all the three-dimensional cultures, hepatocytes appeared morphologically intact without any statistically significant difference in metabolic activity . Collagen-coating did not influence cell attachment to polyester substrates, whereas woven-nonwoven hydrophilic polyester fabric may offer some potential advantages as three-dimensional system for hepatocyte culture in hybrid liver support systems.

Genet Anal, 1999 Nov, 15(3-5), 179 - 87
Transgenic rabbits for the production of biologically-active recombinant proteins in the milk; Castro FO et al.; The use of live bioreactors for the expression of human genes in the mammary gland of transgenic animals is one of the most cost-effective ways for the production of valuable recombinant therapeutic proteins . Among the transgenic species used so far, rabbits are good candidates for the expression of tens to hundreds of grams of complex proteins in the milk during lactation . The lactating mammary gland of rabbits has proven to be effective in the processing of complex proteins . In this work . the potential use of rabbits as bioreactors is discussed based on our results and the published data.

Genet Anal, 1999 Nov, 15(3-5), 155 - 60
Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland; Van Cott KE et al.; The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins . However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain . For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels . At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing . However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity . Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

Immunopharmacology, 1999 Nov, 44(3), 273 - 9
Multiple interleukin-2 signaling pathways differentially regulated by microgravity; Licato LL et al.; Defects in innate immunity have been demonstrated in astronauts after space flight . To investigate the role of microgravity on innate immune function, we evaluated NK and LAK activity of human PBMC stimulated with IL-2 under conditions of simulated microgravity, by using a rotating wall vessel (RWV) culture system . Under these conditions, both NK and LAK activity were generated at levels comparable to those found in static flask cultures . The phenotype of the activated PBMC was similar between the two culture conditions, with one notable exception: the IL-2 receptor alpha chain (CD25), which failed to be upregulated in simulated microgravity . To further investigate this change in IL-2 signaling, we examined the ability of IL-2 to induce secondary cytokines . The production of IFNgamma, IL-1beta, and TNFalpha was almost completely abrogated in the microgravity cultures, suggesting that the IL-2 signaling pathways leading to various IL-2-mediated effects are differentially regulated under bioreactor culture conditions.

J Biotechnol, 1999 Oct 8, 75(2-3), 173 - 85
Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum; White S et al.; The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum . Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis . The culture response was assessed chemically and morphologically, using computerised image analysis . High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis . However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss . Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process . Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P . chrysogenum . By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P . chrysogenum.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Sep-Nov, 27(5-6), 399 - 402
Production of urokinase by HT 1080 human kidney cell line; Roychoudhury PK et al.; Studies were carried out in T-flasks and bioreactor to produce urokinase enzyme using HT 1080 human kidney cell line . While growing the cell line it has been observed that the lag phase is reduced considerably in the bioreactor as compared to T-flask culture . The HT 1080 cell adhesion rate and urokinase production were observed to be the function of serum concentration in the medium . The maximum urokinase activity of 3.1 x 10(-4) unit ml(-1) was achieved in the bioreactor at around 65 h of batch culture . Since HT 1080 is an anchorage dependent cell line, therefore, the hydrodynamic effects on the cell line were investigated.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Sep-Nov, 27(5-6), 393 - 8
Studies on the kinetics of Isopropyl Palmitate synthesis in packed bed bioreactor using immobilized lipase; Kee CY et al.; The objective of this research was to study the kinetics of synthesis of a commercially important ester - Isopropyl Palmitate (IPP) using immobilized lipase (Lipozyme IM) . It was studied in a packed bed differential reactor . In order to establish the kinetics of the reaction, parameters such as linear velocity of the fluid through the reactor, particle size, substrate concentration, substrate molar ratio, temperature and water activity were studied . Operational and storage stability of the enzyme were also assessed . The reaction followed Michaelis-Menton kinetics as observed from the relationship of initial rate of the reaction as a function of substrate concentration . It was found that the optimum substrate concentration was 0.15M palmitic acid and isopropyl alcohol in 1:1 stoichiometric ratio . Inhibition by excess of isopropyl alcohol has been identified . The optimum temperature for the esterification reaction was found to be around 50 degrees C . The activation energy of this process was determined to be 43.67 kJ/mol . The optimum water content was 0.50% . The reaction rates were measured in the absence of any significant external diffusional limitations . Since internal diffusional limitations could not be eliminated, the kinetics observed is only apparent.

Biotechnol Bioeng, 2000 Jan 20, 67(2), 234 - 9
Development of an on-line monitoring system of human keratinocyte growth by image analysis and its application to bioreactor culture; Kino-Oka M et al.; Human keratinocytes were cultured in serum-free medium for the purpose of on-line cell growth monitoring by image analysis . The validity of a process using a newly developed video microscopy system with image analysis for growth-rate monitoring in real time was verified by the measurement of the degree of confluence of keratinocytes in T-flasks and Petriperm dishes . The growth rate of keratinocytes was calculated subsequently from the linear relationship between average degree of confluence and cell concentration . This technique was applied to the culture in the bioreactor "KERATOR" in which a special video microscopy system using a CCD camera was built . The cell concentration evaluated by image analysis agreed well with that evaluated by conventional direct cell counting after enzymatic digestion, and the on-line monitoring of the specific growth rate allowed identification of both lag- and exponential-growth phases of the culture .

Biotechnol Bioeng, 2000 Jan 20, 67(2), 224 - 33
Dynamic modeling and optimal fed-batch feeding strategies for a two-phase partitioning bioreactor; Cruickshank SM et al.; A dynamic model for the degradation of phenol in a two-phase partitioning bioreactor has been developed based on mechanistic balances around the bioreactor . The key process characteristics including substrate transfer between the organic and aqueous phases, substrate inhibition, oxygen limitation, and cell entrainment were incorporated into the model . The model predictions were validated against existing experimental data obtained for a 2-L bioreactor, and good correlation was observed for the time frames of the simulations, as well as for trends in cell and substrate concentrations . Optimal fed-batch, phenol feeding strategies were then developed based on two approaches: (1) maximization of phenol consumption in a fixed time interval and (2) consumption of a fixed amount of phenol in minimal time . The optimal feeding policies, determined using the Iterative Dynamic Programming algorithm, provided substantial improvements in the amount of phenol consumed when compared to a typical experimental heuristic approach . For example, 45.73 g of phenol was predicted to be consumed in 50 h (not including lag phase) using the optimal feeding profile compared to 10.26 g of phenol consumed in the simulated experimental approach . Oxygen limitation was predicted to be a recurring operational challenge in the partitioning bioreactor, and had a strong impact on the optimization results .

Biotechnol Bioeng, 2000 Jan 20, 67(2), 197 - 205
Dynamic optimization of hybridoma growth in a fed-batch bioreactor; Dhir S et al.; This study addressed the problem of maximizing cell mass and monoclonal antibody production from a fed-batch hybridoma cell culture . We hypothesized that inaccuracies in the process model limited the mathematical optimization . On the basis of shaker flask data, we established a simple phenomenological model with cell mass and lactate production as the controlled variables . We then formulated an optimal control algorithm, which calculated the process-model mismatch at each sampling time, updated the model parameters, and re-optimized the substrate concentrations dynamically throughout the time course of the batch . Manipulated variables were feed rates of glucose and glutamine . Dynamic parameter adjustment was done using a fuzzy logic technique, while a heuristic random optimizer (HRO) optimized the feed rates . The parameters selected for updating were specific growth rate and the yield coefficient of lactate from glucose . These were chosen by a sensitivity analysis . The cell mass produced using dynamic optimization was compared to the cell mass produced for an unoptimized case, and for a one-time optimization at the beginning of the batch . Substantial improvements in reactor productivity resulted from dynamic re-optimization and parameter adjustment . We demonstrated first that a single offline optimization of substrate concentration at the start of the batch significantly increased the yield of cell mass by 27% over an unoptimized fermentation . Periodic optimization online increased yield of cell mass per batch by 44% over the single offline optimization . Concomitantly, the yield of monoclonal antibody increased by 31% over the off-line optimization case . For batch and fed-batch processes, this appears to be a suitable arrangement to account for inaccuracies in process models . This suggests that implementation of advanced yet inexpensive techniques can improve performance of fed-batch reactors employed in hybridoma cell culture .

Biotechnol Bioeng, 2000 Jan 20, 67(2), 189 - 96
Considerations for osmolality measurement under elevated pCO(2): comparison of vapor pressure and freezing point osmometry; Schmelzer AE et al.; Osmolality increases with pCO(2) in bioreactors with pH control, and it has been shown that osmolality compensation by decreasing the basal NaCl concentration partially mitigates the adverse effects of elevated pCO(2) on animal cell growth, protein production, and glycosylation . Thus, measurement of osmolality is important for a complete characterization of the culture environment under elevated pCO(2) . However, osmolality measurement may be compromised by CO(2) evolution . Freezing point depression and vapor pressure depression osmometry were directly compared for the measurement of osmolality in samples at elevated pCO(2) (up to 250 mmHg) and at a variety of pH values (6.7-7.5) . More extensive degassing may be expected with the vapor pressure osmometer due to the smaller sample volume and larger surface area employed . However, both types of osmometer yielded similar results for all pCO(2) and pH values studied . Moreover, the measured values agreed with osmolality values calculated using a semi-empirical model . Further analysis showed that, while sample degassing may result in a large decrease in pCO(2), there is little associated decrease in osmolality . The great majority of total CO(2) in solution is present as bicarbonate (HCO(3)(-)) . Although a small amount of HCO(3)(-) is converted to CO(2) to compensate for CO(2) evolution, further depletion of HCO(3)(-) is inhibited by the associated increase in medium pH and by the need for HCO(3)(-) to maintain charge neutrality in solution . This explanation is consistent with the observed similarity in osmolality values for the two types of osmometer . It was also observed that osmolality did not change in samples that were frozen at -20 degrees C for up to 1 year .

Biotechnol Bioeng, 2000 Jan 20, 67(2), 134 - 40
Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter; James RI et al.; The mouse mammary tumor virus (MMTV) promoter is induced by the addition of a glucocorticoid hormone or analog such as dexamethasone . The hormone binds to its specific transcription factor, glucocorticoid receptor (GR), and the activated complex then binds to the glucocorticoid response elements (GREs) in the enhancer region of the MMTV promoter to induce the overexpression of downstream genes . We have constructed an expression vector for a reporter protein, secreted alkaline phosphatase (SEAP),controlled by the MMTV promoter and co-transfected this vector along with a GR expression cassette into Chinese hamster ovary (CHO) cells . High producers were cloned and grown in suspension cultures . A very high titer, over 0.4 mg/mL, of SEAP was obtained from this inducible overexpression system, about ten times that achievable with the same reporter protein using the strong constitutive SV40 promoter in CHO cells . A peak production rate of 187 pg SEAP per cell per day was observed within 3 days after induction, compared to the peak rate of 23 pg SEAP per cell per day expressed using the constitutive SV40 promoter . With the reduced or zero growth rate during the protein production phase, this novel MMTV overexpression system is highly suited for optimizing glycoprotein synthesis rates in high cell density fed-batch or perfusion bioreactors .

Adv Biochem Eng Biotechnol, 2000, 66, 65 - 82
Electronic noses for bioreactor monitoring; Mandenius CF; Electronic noses provide new possibilities for monitor the state of a cultivation non-invasively in real-time . The electronic nose uses an array of chemical gas sensors that monitors the off-gas from the bioreactor . By taking advantage of the off-gas components' different affinities towards the sensors in the array it is possible with the help of pattern recognition methods to extract valuable information from the culture in a way similar to the human nose . For example, with artificial neural networks, metabolite and biomass concentration can be predicted, the fermentability of a medium before starting the fermentation estimated, and the growth and production stages of the culture visualized . In this review these and other recent results with electronic noses from monitoring microbial and cell cultures in bioreactors are described.

Dtsch Tierarztl Wochenschr, 1999 Oct, 106(10), 425 - 32
{Transgenic animals as bioreactors in the service of the pharmaceutical industry}; Honscha W; A modern pharmacotherapy without the use of proteins as drugs is not more practicable . In the clinic blood coagulation factors (factor VIII and IX), growth hormones (human growth hormone), enzymes (1-antitrysin, insulin), and cytokines are currently used . At the beginning, the proteins were isolated from biological sources or expressed in vitro by the use of E . coli or eukaryotic cell lines . At the moment efforts were undertaken to express these proteins in the milk of transgenic animals . This review article describes the methods for the generation of transgenic animals, the benefits and drawbacks of this new technique in comparison to established methods for the production of proteins as pharmaceuticals . At the end of the review possible improvements of the method are described.

Biotechnol Prog, 1999 Nov-Dec, 15(6), 1133 - 41
Rapid calibration of near-infrared spectroscopic measurements of mammalian cell cultivations; Riley MR et al.; Near-infrared (NIR) spectroscopy is a flexible method that can be employed to noninvasively monitor the concentrations of multiple nutrients and wastes in mammalian cell bioreactors . Development of suitable calibrations can be a labor- and time-intensive process that must be repeated when process conditions are altered significantly . To address this difficulty, we have produced a new approach for generating NIR spectroscopic calibrations that requires significantly less time compared with standard calibration schemes . This method reduces development time from the present level of several weeks to several hours . A small number of experimentally collected spectra serve as inputs to a computational procedure that yields a large number of simulated spectra, each containing both analyte-specific and analyte-independent information . Such simulated spectra may be employed as a calibration set for quantifying analytes in experimentally collected spectra . Spectroscopic measurements of the concentrations of five components (ammonia, glucose, glutamate, glutamine, and lactate) can be accomplished with levels of error similar to those obtained with full experimental calibrations . A key to this process is the utilization of random numbers, which randomizes the influence of natural variations, present in each experimentally collected spectrum, on the resultant composite spectrum . This approach may increase the feasibility of employing NIR spectroscopy to monitor bioreactors and other biological processes subjected to varying operating conditions.

Biotechnol Prog, 1999 Nov-Dec, 15(6), 1065 - 71
Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen; Hu YC et al.; Structural proteins of the poultry pathogen, infectious bursal disease virus (IBDV), were expressed in the baculovirus/insect cell expression system . To date, several reports have indicated that animal virus structural proteins are expressed only at low yield in this system . In this article, several factors were examined to enhance yield . These include medium, dissolved oxygen level, and the addition (in vivo and in vitro) of protease inhibitors . Specifically, two media were compared, and SF-900 II was superior to Ex-Cell 401 for cell growth and IBDV protein expression . A cocktail of protease inhibitors including phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and ethylenediamine tetraacetic acid (EDTA) minimized proteolysis in vitro . Also, aprotinin and pepstatin A deterred product degradation in vivo and increased the product yield nearly 2-fold . Finally, in 3 L bioreactors, a dissolved oxygen tension of 50% DO (air saturation) was optimal . Results demonstrated that several relatively simple adjustments to the baculovirus system significantly improved the yield of IBD virus structural proteins.

Int J Artif Organs, 1999 Oct, 22(10), 701 - 9
Significantly improved survival time in pigs with complete liver ischemia treated with a novel bioartificial liver; Flendrig LM et al.; Aim of the study was to evaluate treatment efficacy and safety of a scaled-up version of our porcine hepatocytes based BAL system in pigs with complete liver ischemia (LIS) . Thirty-one pigs underwent total devascularization of the liver (LIS) by termino-lateral porta-caval shunts and sutures around the bile duct, the common hepatic and gastroduodenal arteries and their accessory branches . The hepato-duodenal ligament was completely transected . Four experimental groups were studied: the first control group (LIS Control, n = 10) received glucose infusion only, the second control group (LIS Plasmapheresis, n = 8) was connected to a centrifugal plasma-separator with a bottle representing the bioreactor volume, the third control group (LIS Empty-BAL, n = 5) received BAL treatment without cells, and the treated group (LIS Cell-BAL, n = 8) was connected for a maximum period of 24 hours to our scaled-up BAL seeded with around 14 billion viable primary porcine hepatocytes . BAL treatment significantly prolonged life in large animals (approximately 35 kg) with complete LIS (Controls, mean +/- SEM: 33.1 +/- 3 h, Cell-BAL: 51.1 +/- 3.4 h; p = 0.001; longest survivor 63 h) . In addition, blood ammonia and total bilirubin levels decreased significantly, indicating metabolic activity of porcine hepatocytes in the bioreactor . No significant differences were noticed among the three control groups, indicating that there was no device effect and that the plasmapheresis procedure was well tolerated . No important adverse effects were observed.

Hum Gene Ther, 1999 Nov 20, 10(17), 2799 - 810
Combined ultrafiltration-transduction in a hollow-fiber bioreactor facilitates retrovirus-mediated gene transfer into peripheral blood lymphocytes from patients with mucopolysaccharidosis type II; Pan D et al.; The process of growing and transducing large quantities of human primary peripheral blood lymphocytes (PBLs) with high gene transfer efficiency continues to be one of the major challenges for clinical and experimental gene therapy . Toward developing a clinical trial of lymphocyte gene therapy for mucopolysaccharidosis type II (i.e., Hunter syndrome), we investigated a novel method that exploited the innate capability of a hollow-fiber bioreactor system to filter large quantities of vector supernatant and facilitate transduction . An aliquot (5 x 10(7)) of PBL apheresis product was precultured in a gas-permeable culture bag or a bioreactor, and then transduced with a retroviral vector L2SN containing the iduronate-2-sulfatase (IDS) and neomycin resistance genes . We observed that the total number of PBLs could be expanded up to 187-fold, yielding up to 10(10) cells at the end of a 7-day culture period . The multiplicity of infection could be increased (up to 20-fold) by ultrafiltrating a large volume of vector supernatant through the semipermeable membrane of this system . A high level of transduction efficiency (up to 57%) was achieved, resulting in IDS enzyme activity as high as 1250 U/mg/hr in transduced PBL(MPS) 15 days after transduction . This level was markedly increased from that of nontransduced cells (<3 U/mg/hr) and was even greater than that of normal PBLs (mean, 809; n = 10) . After 12 days of G418 selection, PBL(MPS) transductants exhibited a proviral IDS enzyme level approximately threefold higher than that in normal PBLs . These results indicated that the hollow-fiber bioreactor could be used to culture and transduce human primary PBLs in clinically useful quantities with relatively high gene transfer efficiency and transgene expression.

Microbios, 1999, 99(394), 147 - 59
Effect of nitrogen source on pullulan production by Aureobasidium pullulans grown in a batch bioreactor; West TP et al.; Pullulan production by Aureobasidium pullulans ATCC 201253 using selected nitrogen sources was studied in a medium using corn syrup as a carbon source . Independent of the corn syrup concentration present, the use of corn steep liquor or hydrolysed soy protein as a nitrogen source instead of ammonium sulphate did not elevate polysaccharide production by ATCC 201253 cells grown in an aerated, batch bioreactor containing 4 litres of medium . Pullulan production on corn steep liquor or hydrolysed soy protein as a nitrogen source became more comparable as the concentration of corn syrup was increased . Cell weights after 7 days of growth on any of the nitrogen sources were similar . The viscosity of the polysaccharide on day 7 was highest for cells grown on ammonium sulphate and 12.5% corn syrup . The pullulan content of the polysaccharide elaborated by ammonium sulphate-grown cells on day 7 decreased as the corn syrup level rose in the medium while the pullulan content of polysaccharide produced by cells grown on corn steep liquor or soytone generally increased.

Cell Transplant, 1999 Sep-Oct, 8(5), 531 - 41
A new bioartificial liver using porcine hepatocyte spheroids in high-cell-density suspension perfusion culture: in vitro performance in synthesized culture medium and in 100% human plasma; Sakai Y et al.; A prototype of a bioartificial liver (BAL) based on suspension perfusion culture of porcine hepatocyte spheroids was developed at 150 ml scale . About 2% (4 x 10(9) cells) of whole human liver cells was immobilized . The cell density in the bioreactor was 2.7 x 10(7) cells/ml, which was almost comparable to that of presently developed packed-bed-type BALs . The bioreactor was perfused with culture medium while retaining spheroids . This was done using a rotating stainless filter (pore size 50 microm) . In vitro 8-h perfusion experiments utilizing both synthesized culture medium and 100% human plasma demonstrated the spheroids in the bioreactor had almost the same functions on a unit/cell basis as those in small-scale rotational culture . This indicated that the functional deterioration often associated with scaling up had been minimized . Rapid spheroid aggregation and dysfunction in specific human plasma pool must be eliminated before clinical application, although this phenomenon seemed to be inherent to porcine hepatocyte-based BALs . This prototype shows promise in meeting present clinical demands by achieving maximal metabolic activities even in the short term.

Biotechnol Bioeng, 1999, 66(3), 171 - 9
Control of pH in large-scale, free suspension animal cell bioreactors: alkali addition and pH excursions; Langheinrich C et al.; It is likely that, in the future, animal cell cultures of a higher cell density and/or cell lines with higher specific oxygen demands will be available . Such developments will lead to the need for improved homogeneity in the bioreactor and a greater supply of nutrients . The accompanying significant increase in CO(2) production and accumulation and the resulting reduction in pH are also important implications for process engineering . Such pH reduction is typically controlled by the addition of sodium carbonate . Previous studies using flow visualization mimicking the operating conditions in a typical plant-scale reactor showed potentially cell-damaging regions within it due to pH excursions . This paper confirms the existence of these excursions by pH measurements in the alkali addition zone . It also identifies the accumulation of alkali in a region of poor local liquid homogenization as a serious scale-up problem and shows how a change in the addition point significantly reduces it .

Clin Cancer Res, 1999 Oct, 5(10 Suppl), 3101s - 3105s
Generation and monitoring of cell lines producing humanized antibodies; Losman MJ et al.; Antibody humanization has eliminated or reduced the human antimouse antibody response associated with the administration of murine antibodies . We have successfully humanized three different antibodies: (a) hMN-3 (granulocyte targeting); (b) hMu-9 (colorectal cancer targeting); and (c) hWI2 (anti-idiotype to the anti-carcinoembryonic antigen antibody MN-14) . All humanized antibodies demonstrated immunoreactivities comparable to their parent counterparts . Previously, we reported the generation of high productivity cell lines for hMN-14 and hLL2 using the amplifiable vector pdHL2 . Through amplification, selection, and cloning procedures, cell lines capable of large scale production were established, and further enhancement of production was achieved by a fed-perfusion bioreactor process . Using a similar and improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the concentration of methotrexate in the culture media . A reliable IgG determination method is essential to monitor amplification, especially at the final cloning stage, for the selection of the subclones with the highest productivity . We found that measurement of humanized IgG concentration in culture media supplemented with more than 1 microM methotrexate by a standard ELISA assay could be unreliable and misleading . Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and reproducible, the method is time-consuming, tedious, and labor intensive . We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures . The assay is simple and sensitive, and it makes simultaneous determinations of relative antibody production from individual clones at the 96-well stage feasible . With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell lines can be monitored in an efficient manner.

Biotechnol Appl Biochem, 1999 Dec, 30 ( Pt 3), 245 - 50
Immobilized lipoxygenase in a packed-bed column bioreactor: continuous oxygenation of linoleic acid; Hsu AF et al.; The continuous oxygenation of linoleic acid (LA) by immobilized lipoxygenase (LOX) was studied . Enzymatic oxidation was carried out in a recirculating packed column reactor using immobilized LOX as the stationary phase and LA as the substrate . The column, when packed with LOX immobilized in either a calcium alginate sol-gel matrix or a phyllosilicate sol-gel matrix, is equivalent to five continuous stirred tank reactors (CSTRs) . The reactor cascade was calculated from the residence-time distribution for the reactor . Based on mass-balance calculations, a set of mathematical equations for predicting the concentration of oxygenated product generated in each CSTR was calculated . Product formation in the packed column reactor was simulated and results calculated with the model were compared with the experimental results . The data indicated that product yield (hydroperoxyoctadecadienoic acid, HPOD) increased asymptotically with reaction time . Experimentally, when the bioreactor was packed with calcium alginate sol-gel-immobilized LOX, an initial linear increase in HPOD production with time was observed, but reached a steady state . For the bioreactor packed with phyllosilicate sol-gel-immobilized LOX, initial HPOD production increased more rapidly but reached a lower steady-state concentration . From these data, a simple computer simulation model was developed to determine the process kinetics of this reactor design.

Biochem J, 1999 Dec 1, 344 Pt 2, 297 - 303
Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies; Juntunen K et al.; We have expressed recombinant human vitamin D receptor and its ligand-binding domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor . Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l . Gel-filtration analyses indicated that the purified human vitamin D receptor and its ligand-binding domain were present as monomers in solution . The purified vitamin D receptor and its ligand-binding domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM . Neutron scattering studies of the ligand-binding domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides . The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies . The results indicate that we can produce biologically active human vitamin D receptor and its ligand-binding domain in insect cells and purify them for functional and structural studies.

Dev Biol Stand, 1999, 101, 57 - 64
Membrane-based cell culture systems--an alternative to in vivo production of monoclonal antibodies; Nagel A et al.; A new generation of membrane-based cell culture devices especially designed for small scale production of monoclonal antibodies (mabs) has entered the market in the last few years . In contrast to conventional perfusion hollow fibre bioreactors, these devices contain two functionally different membranes--one ultrafiltration membrane for nutrient supply and one gas-permeable membrane for direct oxygenation of cells . The latest systems of this generation are static culture systems which are of moderate cost and either better than, or equal to, the ascites mice in terms of quality and quantity of produced monoclonal antibodies . We have investigated the advantages of the perfused Tecnomouse bioreactor and the static CELLine culture flasks in comparison to ascites production and conventional roller bottle cultures.

Int J Food Microbiol, 1999 Oct 1, 51(1), 39 - 51
Introducing optimal experimental design in predictive modeling: a motivating example; Versyck KJ et al.; Predictive microbiology emerges more and more as a rational quantitative framework for predicting and understanding microbial evolution in food products . During the mathematical modeling of microbial growth and/or inactivation, great, but not always efficient, effort is spent on the determination of the model parameters from experimental data . In order to optimize experimental conditions with respect to parameter estimation, experimental design has been extensively studied since the 1980s in the field of bioreactor engineering . The so-called methodology of optimal experimental design established in this research area enabled the reliable estimation of model parameters from data collected in well-designed fed-batch reactor experiments . In this paper, we introduce the optimal experimental design methodology for parameter estimation in the field of predictive microbiology . This study points out that optimal design of dynamic input signals is necessary to maximize the information content contained within the resulting experimental data . It is shown that from few dynamic experiments, more pertinent information can be extracted than from the classical static experiments . By introducing optimal experimental design into the field of predictive microbiology, a new promising frame for maximization of the information content of experimental data with respect to parameter estimation is provided . As a case study, the design of an optimal temperature profile for estimation of the parameters D(ref) and z of an Arrhenius-type model for the maximum inactivation rate kmax as a function of the temperature, T, was considered . Microbial inactivation by heating is described using the model of Geeraerd et al . (1999) . The need for dynamic temperature profiles in experiments aimed at the simultaneous estimation of the model parameters from measurements of the microbial population density is clearly illustrated by analytical elaboration of the mathematical expressions involved on the one hand, and by numerical simulations on the other.

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