ROLE OF BIOREACTOR IN BIOPROCESS ENGG.-04

Extremophiles, 1999 Aug, 3(3), 199 - 204
A microfiltration bioreactor to achieve high cell density in Sulfolobus solfataricus fermentation; Schiraldi C et al.; A novel technique is proposed to achieve higher cell yield in extremophile fermentation . Because the accumulation of toxic compounds is thought to be responsible for low biomass yields, a bioreactor has been designed based on a microfiltration hollow-fiber module located inside the traditional fermentation vessel . Using the cultivation of the thermoacidophilic archeon Sulfolobus solfataricus theta as a model, a biomass of 35gl(-1) dry weight was obtained which proved greater than that of 2gl(-1) obtained in batch fermentation . The bioreactor was characterized by running several fermentation experiments to check the high stability of the membrane module to sterilization cycles, high temperatures, and acidic pHs, even for prolonged periods of time . It was shown that the exhaust medium is unable to sustain growth for the presence of toxic compounds, and ultrafiltration and ion-exchange techniques were used in all the attempts to regenerate it . The results demonstrated the ability of the method to lower inhibitor concentrations and prolong the growth phase, thus achieving high cell density . Furthermore, they indicated that the toxic compounds are ionic species of less than 1kDa.

Planta Med, 1999 Aug, 65(6), 527 - 31
Biological properties of ulvan, a new source of green seaweed sulfated polysaccharides, on cultured normal and cancerous colonic epithelial cells; Kaeffer B et al.; Ulvans (from Ulva lactuca) constitute a dietary fiber structurally similar to the mammalian glycosaminoglycans but with unexplored biological or cytotoxic activities . From native low-viscosity preparations containing 33.5 molar % and 18.4 molar % of sulfate residues and uronic acid residues, respectively, we derived desulfated, reduced and desulfated-reduced polysaccharides with respectively 5.2, 2.9, and 4.5-4.9 molar % of sulfate residues and uronic acid residues . The effects of these preparations were examined on the adhesion, proliferation and differentiation of normal or tumoral colonic epithelial cells cultured in conventional (0.3-0.8 x 10(6) cells/ml) or rotating bioreactor (3-8 x 10(6) cells/ml) culture conditions . In conventional culture conditions, ulvan modified the adhesion phase and the proliferation of normal colonic cells and undifferentiated HT-29 cells according to their molecular weights and to the relative molar proportion of sulfate residues . From the native polysaccharides, we have screened sulfated ulvans (MW < 5,000) which inhibited the Caco-2 cell proliferation/differentiation program by inducing a low cell reactivity to Ulex europeaus-1 lectins in defined (p < 0.001) or serum-supplemented media (p < 0.01) but were inactive on normal colonocytes . In conclusion, this dietary fiber could be a source of oligosaccharides with a bioactivity, a cytotoxicity or a cytostaticity targeted to normal or cancerous epithelial cells.

J Biotechnol, 1999 May 28, 71(1-3), 105 - 31
Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures; Jones KD et al.; Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol . In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH . We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways . This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.

J Biotechnol, 1999 May 28, 71(1-3), 7 - 16
Computing operating diagrams of bioreactors; Pavlou S; The operating diagram of a bioreactor is an illustrative way to present the effect that the operating conditions have on its long-term behavior . It can be constructed if a mathematical model of the bioreactor is available . The procedure for constructing the operating diagram consists in analyzing the dynamic behavior of the system of the differential equations of the model . Some methods are described that can be used in computing operating diagrams of bioreactors . They are based on numerical bifurcation techniques for systems of differential equations . Both cases of bioreactors with constant and periodically varying operating conditions are considered.

J Gastroenterol Hepatol, 1999 Aug, 14(8), 757 - 70
Extracorporeal support and hepatocyte transplantation in acute liver failure and cirrhosis; Riordan SM et al.; The relative shortage of donor organs and lack of immediate availability mean that many patients with acute liver failure die before orthotopic liver transplantation can be performed . An effective temporary liver support system could improve the chance of survival with or without a transplant being ultimately carried out . Recent technological advances resulting in improved maintenance of hepatocyte viability and function in culture and bioreactor designs which facilitate adequate perfusion of the cellular component and removal of products of cellular metabolism have led to the development of a number of bioartificial devices for liver support . Three such devices have undergone preliminary clinical evaluation in the setting of acute liver failure, with a statistically significant reduction in raised intracerebral pressure along with improvements in consciousness level and some biochemical parameters associated with treatment with one of these . Several other devices with different characteristics have shown promise in vitro and/or in animal models but await clinical evaluation . Several new totally artificial systems have also been described, along with the emergence of isolated hepatocyte transplantation, with reports of successful 'bridging' to liver transplantation. h, k, a, e, g. Controlled trials on a multicentre basis in well-defined patient groups and with standardized outcome measures will be required to properly evaluate the clinical value of each of these approaches to providing liver support in acute liver failure and cirrhosis . A better understanding of mechanisms underlying multiorgan failure and of factors inhibiting liver regeneration, thereby allowing a more targeted approach, will be essential to the further development of effective liver support strategies in these settings.

Trends Biotechnol, 1999 Oct, 17(10), 389 - 95
Colloidal gas aphrons: potential applications in biotechnology
Jauregi P, Varley J.
Colloidal gas aphrons are microbubbles encapsulated by surfactant multilayers . They provide a large interfacial area to adsorb charged and/or hydrophobic molecules; the extent and mechanism of the adsorption depends on the surfactant multilayer . The physical properties of colloidal gas aphrons have recently been characterized for a range of surfactants in order to find the best systems for particular applications . A range of exciting biotechnology applications has been identified, including the recovery of cells, proteins and other biological molecules, and the enhancement of gas transfer in bioreactors and bioremediation.

Transgenic Res, 1999 Jun, 8(3), 237 - 47
Expression of human blood clotting factor VIII in the mammary gland of transgenic sheep; Niemann H et al.; By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'bioreactor' for producing pharmacologically active proteins on a large scale . Here we report the generation of transgenic sheep bearing a fusion gene construct with the human blood clotting factor VIII (hFVIII) cDNA under the transcriptional control of a 2.2 kb fragment of the mammary gland specific promoter of the ovine beta-Lactoglobulin (beta-Lac) gene . Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine metallothionein (MtI) gene (beta-Lac/hFVIII-MtI) . Founders transmitted the transgene in a Mendelian fashion and two transgenic lines were generated . Ten out of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk . But only in transgenic F1 ewes 4010 and 603 hFVIII clotting activity estimated at 4-6 ng/ml was detected in defatted milk . Furthermore, the presence of rhFVIII-protein in ovine milk was demonstrated by a specific band at approximately 190 kD following immunoprecipitation and immunoblotting . Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesting that the employed beta-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland . A rhFVIII standard preparation (rhFVIIIstd) was rapidly sequestered in a saturable fashion in ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained . Recovery of hFVIIIstd was dependent on milk donor, storage temperature and dilution of milk sample.

Ann Biomed Eng, 1999 Jul-Aug, 27(4), 572 - 9
Estimation of the shear stress on the surface of an aortic valve leaflet; Weston MW et al.; The limited durability of xenograft heart valves and the limited supply of allografts have sparked interest in tissue engineered replacement valves . A bioreactor for tissue engineered valves must operate at conditions that optimize the biosynthetic abilities of seeded cells while promoting their adherence to the leaflet matrix . An important parameter is shear stress, which is known to influence cellular behavior and may thus be crucial in bioreactor optimization . Therefore, an accurate estimate of the shear stress on the leaflet surface would not only improve our understanding of the mechanical environment of aortic valve leaflets, but it would also aid in bioreactor design . To estimate the shear stress on the leaflet surface, two-component laser-Doppler velocimetry measurements have been conducted inside a transparent polyurethane valve with a trileaflet structure similar to the native aortic valve . Steady flow rates of 7.5, 15.0, and 22.5 L/min were examined to cover the complete range possible during the cardiac cycle . The laminar shear stresses were calculated by linear regression of four axial velocity measurements near the surface of the leaflet . The maximum shear stress recorded was 79 dyne/cm2, in agreement with boundary layer theory and previous experimental and computational studies . This study has provided a range of shear stresses to be explored in bioreactor design and has defined a maximum shear stress at which cells must remain adherent upon a tissue engineered construct.

Biotechnol Bioeng, 1999 Oct 20, 65(2), 219 - 26
Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase in Escherichia coli; Chou CP et al.; A novel and simple method of using penicillin for screening of mutant strains with a high penicillin acylase (PAC) activity was developed . Random mutagenesis was conducted using a PAC-producing strain resistant to 6-aminopenicillanic acid (6-APA) as the parent strain and mutants were screened with penicillin at a high concentration . Results suggest that mutants with a high minimum inhibitory concentration for penicillin (MIC(penG)) usually overproduce PAC . Both volumetric and specific PAC activities of a mutant, MD7, were significantly higher than those of the parent strain, HBPAC101 harboring pCLL2902 . The mutation(s) resulting in the enhanced expression was mapped on the host chromosome rather than the plasmid . In addition, the mutant strain of MDDeltaP7, derived by elimination of the harbored plasmid in MD7, was demonstrated to be efficient in production of PAC by using the expression plasmids for which expression of the pac gene is limited by translation . An extremely high specific PAC activity of more than 350 U/L/OD(600) was reached upon cultivation of MDDeltaP7 harboring pTrcKnPAC2902 in a bioreactor . As such, the strategy is effective in terms of constructing PAC overproducers and improving the process yield for production of PAC .

J Vet Med Sci, 1999 Jul, 61(7), 861 - 3
Bioremediation of trichloroethylene and cis-1,2-dichloroethylene-contaminated groundwater by methane-utilizing bacteria; Arai K et al.; Experimental studies on the bioremediation of groundwater contaminated with low concentration trichloroethylene (TCE) and cis1,2-dichloroethylene (DCE) were performed with two sets of bioreactors . Reactors No . 1 and No . 2 were operated without and with methane supplement, respectively . No inoculum was used . The concentrations of TCE and DCE in the effluent and the off gas from reactor No . 2 were much lower than those from reactor No . 1 . When air and an H2O2 solution were supplied to reactor No . 2, concentrations of TCE and DCE in the effluent and the off gas were lower than the lowest detectable limit . The population of methane-utilizing bacteria in reactor No . 2 was 1,000 times higher than that in groundwater or in the effluent from reactor No . 1 . These methane-utilizing bacteria were apparently attributable to the treatment of TCE.

J Ind Microbiol Biotechnol, 1999 Jul, 23(1), 691 - 6
Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis; Zhang XW et al.; High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode . A high cell density of 2.65 g x L(-1) (batch culture) or 2.74 g x L(-1) (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg x L(-1)) was about 20.5% higher than in the batch culture (53.43 mg x L(-1)) . An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed . Good agreement was found between the model predictions and experimental data . The models demonstrated that the optimal light intensity for mixotrophic growth of H . pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90-360 micromol x m(-2) x s(-1), and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode.

Curr Opin Biotechnol, 1999 Aug, 10(4), 370 - 5
Applications of oxidoreductases; May SW; Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions . Because many chemical and biochemical transformations involve oxidation/reduction processes, developing practical biocatalytic applications of oxidoreductases has long been an important goal in biotechnology . During the past year, significant progress has been made in the development of oxidoreductase-based diagnostic tests and improved biosensors, in the design of innovative systems for regeneration of essential coenzymes, in the construction bioreactors for biodegradation of pollutants and for biomass processing, and in the development of oxidoreductase-based approaches for synthesis of polymers and oxyfunctionalized organic substrates.

Exp Toxicol Pathol, 1999 Jul, 51(4-5), 432 - 5
Use of rat hepatocytes immobilized in agarose gel threads for biosynthesis of metabolites of potential cytostatics; Trejtnar F et al.; The aim of this work was to evaluate possibility of use of the rat isolated hepatocytes immobilized in agarose gel and continuously perfused for production of needed metabolites of two potential cytostatics, benfluron (5-(2-dimethylamino-ethoxy)-7-oxo-7H-benzo{c}fluorene) and oracin (6-{2-(hydroxyethyl) amino-ethyl}-5,11-dioxo-5,6-dihydro-11H-indeno {1,2-c}isoquinoline) . The rat isolated hepatocytes obtained by two-step collagenase perfusion method were immobilized in agarose threads and perfused in a small bioreactor under a recirculation regimes . Biosynthesis of 9-hydroxybenfluron and 3-hydroxyoracin in immobilized rat hepatocytes was studied . Yields of the metabolites of interest in hepatocytes in immobilized and perfused rat hepatocytes was compared to production of metabolites in hepatocyte suspension and in rats in vivo . 9-hydroxybenfluron was presented during perfusion of immobilized rat hepatocytes in a relatively high amounts but total recovery all forms of benfluron was very low due to especially high binding to components of the perfusion system . More effective method remains the production of 9-hydroxybenfluron in rats in vivo . A considerable biosynthesis of 3-hydroxyoracin by immobilized rat hepatocytes in the bioreactor was found . Concentration of the metabolite in the perfusate rose continuously during 6 hours of perfusion . 3-hydroxyoracin production was increased several times with use of immobilized hepatocytes from rats treated for three days with methylcholanthrene . The yield of 3-hydroxyoracin in rats in vivo was comparably high but an advantage of in vitro synthesis is a much shorter interval to obtain the same amount of the metabolite of interest . In spite of some limitations in compounds exerting high trapping in the perfusion system, the method of the immobilized and perfused hepatocytes can be very useful and effective for production of some drug metabolites in biochemistry and pharmacology.

Am J Physiol, 1999 Aug, 277(2 Pt 2), H433 - 44
Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies; Bursac N et al.; The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies . Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively . After 1 wk, all constructs contained a peripheral tissue-like region (50-70 micrometer thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration . Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles . Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale . Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles . These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

Biotechnol Prog, 1999 Jul-Aug, 15(4), 630 - 9
Development and validation of a methodology for intracellular pH measurements of hybridoma cells under bioreactor culture conditions; Cherlet M et al.; The intracellular pH (pH(i)) is an important factor in the regulation of different cellular processes . It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment . We developed and validated therefore a methodology that permits a reproducible and reliable pH(i) measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions . The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM (BCECF = 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), and analyzed by flow cytometry . Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer . All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pH(i) value measured . Additional experiments were carried out, showing the representativeness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.

Biotechnol Bioeng, 1999 Oct 5, 65(1), 44 - 53
The effect of temperature on the continuous ferrous-iron oxidation kinetics of a predominantly Leptospirillum ferrooxidans culture; Breed AW et al.; The ferrous-iron oxidation kinetics of a bacterial culture consisting predominantly of Leptospirillum ferrooxidans were studied in continuous-flow bioreactors . The bacterial culture was fed with a salts solution containing 12 g/L ferrous-iron, at dilution rates ranging from 0.01 to 0.06 l/h, and temperatures ranging from 30 to 40 degrees C, at a pH of 1.75 . The growth rate, and the oxygen and ferrous-iron utilization rates of the bacteria, were monitored by means of off-gas analysis and redox-potential measurement . The degree-of-reduction balance was used to compare the theoretical and experimental values of r(CO(2)), -r(O(2)) and -r(Fe(+2)), and the correlation found to be good . The maximum bacterial yield on ferrous-iron and the maintenance coefficient on ferrous-iron, were determined using the Pirt equation . An increase in the temperature from 30 to 40 degrees C did not appear to have an effect on either the maximum yield or maintenance coefficient on ferrous-iron . The average maximum bacterial yield and maintenance coefficient on ferrous-iron were found to be 0.0059 mmol C/mmol Fe(2+) and 0.7970 mmol Fe(2+)/mmol C)/h, respectively . The maximum specific growth rate was found to be 0.077 l/h . The maximum specific ferrous-iron utilization rate increased from 8.65 to 13.58 mmol Fe(2+)/mmol C/h across the range from 30 to 40 degrees C, and could be described using the Arrhenius equation . The kinetic constant in bacterial ferrous-iron oxidation increased linearly with increasing temperature . The ferrous-iron kinetics could be accurately described in terms of the ferric/ferrous-iron ratio by means of a Michaelis-Menten-based model modified to account for the effect of temperature . A threshold ferrous-iron level, below which no further ferrous-iron utilization occurred, was found at a ferric/ferrous-iron ratio of about 2500 . At an overall iron concentration of 12 g/L, this value corresponds to a threshold ferrous-iron concentration of 78.5 x10(-3) mM .

Biotechnol Bioeng, 1999 Oct 5, 65(1), 17 - 23
Immobilization of recombinant heparinase I fused to cellulose-binding domain; Shpigel E et al.; Immobilization of biologically active proteins is of great importance to research and industry . Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins . We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD . Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD) . The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies . The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column . The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin . C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose . CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity . By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled . The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared . The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization . Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step .

Zhonghua Nei Ke Za Zhi, 1997 Oct, 36(10), 665 - 8
{Establishment and significance of an extracorporeal bioartificial liver support system}; Wang Y et al.; This experiment was carried out to establish an optimal extracorporeal bioartificial liver support system (EBLSS) and furnish a new therapy for hepatic failure . For this purpose, human hepatocytes were isolated by two-stage perfusion method and cultured by spheroidal aggregates method . Besides, hollow fiber tube was used as bioreactor and the hemodialysis instrument was refited to serve as supplementary circulation unit, which was composed of EBLSS and was tried in normal dogs . A total of 2 x 10(10) human hepatocytes with a viability of 94% was obtained . These hepatocytes became multicellular spheroidal aggregates under non-adherent conditions . After 5 hours circulating on EBLSS, the hepatocytes remained active . At the same time, the monitoring system revealed a normal vital sign in dogs and the laboratory tests found their serum total protein and albumin were increased . These results show that the key technology of bioartificial liver has been dereloped basically and this EBLSS is effective and safe, providing a foundation for the support and treatment of hepatic failure with bioartificial liver.

Ther Apher, 1999 Aug, 3(3), 264 - 7
A new concept of bioartificial liver based on a fluidized bed bioreactor; Dore E et al.; Many bioartificial livers have been developed, but most of them suffer from difficulty when being scaled up and from poor efficiency of mass transfer between the plasma and the immobilized hepatocytes . We present a new concept of bioartificial liver based on the fluidized bed motion of hepatocytes entrapped in alginate beads . The bioreactor is designed to offer stable behavior . The maximum fluid perfusion velocity is determined to avoid any bead release from the bioreactor . The fluidized bed height depends on the amount of beads and the velocity employed . Under the optimized operating conditions, the mass transfer between perfusion fluid and beads is very efficient; only 10 min are necessary to reach concentration equilibrium . Hence, this fluidized bed bioartificial liver appears to be a promising tool for a liver support system in the treatment of acute liver failure.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Jul, 27(4), 313 - 42
Dual enzyme multi-layer bioreactors: analytical modeling and experimental studies; Segal V et al.; Enzymic reactors are developed for a variety of biomedical-biotechnological applications, including blood detoxification . For the latter, an appropriate approach is to use enzymes of the Mercapturic Acid Pathway . The first two enzymes of this pathway are Glutathione-S-Transferase (GST) and gamma-Glutamyl Transpeptidase (gamma GT) . Earlier, the performance of an immobilized GST reactor was investigated experimentally and theoretically . Here, the analytical model was extended to describe a dual-enzyme continuous packed-bed reactor (DCP), in which the two enzymes (E1 and E2) are arranged in alternating layers . The performance of DCP reactors was first studied by numerical simulations, considering the effects of reactor configuration (i.e . number of enzyme layers), kinetic characteristics (K(m), Vmax, Kiq) and operational parameters (flow rate, substrates concentration) . Results were obtained in terms of substrate and products concentration profiles along the reactor . The theoretical calculation were supplemented by experimental studies . In the latter GST (i.e . E1) and gamma GT (i.e . E2), were used when immobilized on porous beads, and the reactor was set up and operated in various configurations . It was found that the factors which mostly affect the performance of DCP systems are reactor configuration and extent of inhibition of E1 by its reaction product.

Int J Oncol, 1999 Sep, 15(3), 511 - 8
Production of ex vivo expanded hematopoietic cells and progenitors in a closed bioreactor, starting with a small volume marrow collection: A feasibility study in patients with poor-risk breast cancer and receiving high-doses of cyclophosphamide; Chabannon C et al.; We report a clinical pilot study conducted in 6 women with poor-prognosis breast cancer . The goal was to evaluate the feasibility and safety of producing hematopoietic progenitors and cells from a small marrow sample, for clinical use after high-dose cyclophosphamide . A small volume marrow collection was obtained, using local anesthesia and conscious sedation, before the first of two chemotherapy cycles . Cells were cryopreserved, and later thawed to inoculate two Aastrom Biosciences Inc Replicell bioreactors, on time to reinfuse ex vivo expanded cells after the second chemotherapy cycle . Patients recovered neutrophils and platelets at similar times after the first and second chemotherapy cycles, and showed comparable clinical events . This pilot study prepares future randomized trials, designed to evaluate clinical benefits associated with the use of ex vivo expanded cells in the setting of multicycle high-dose chemotherapy.

Appl Environ Microbiol, 1999 Aug, 65(8), 3750 - 3
Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum; Gabler M et al.; The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1 . 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source . The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers . The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass) . The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase . Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer . Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

Appl Environ Microbiol, 1999 Aug, 65(8), 3265 - 71
Engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates; Prieto MA et al.; In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans . We used the mini-Tn5 system as a tool to construct Escherichia coli 193MC1 and P . oleovorans POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection . The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level . E . coli 193MC1 produced considerably shorter polyesters than P . oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7 . Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution . In addition, we found that E . coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.


Moisture content is the single most important factor that promotes the accelerated decomposition. The bioreactor technology relies on maintaining optimal moisture content near field capacity (approximately 35 to 65%) and adds liquids when it is necessary to maintain that percentage. The moisture content, combined with the biological action of naturally occurring microbes decomposes the waste. The microbes can be either aerobic or anaerobic. A side effect of the bioreactor is that it produces landfill gas (LFG) such as methane in an anaerobic unit at an earlier stage in the landfill’s life and at an overall much higher rate of generation than traditional landfills.

In batch cultivation, an innoculum of known density is seeded into a specified volume of pre-conditioned medium in the bioreactor. Ideally nothing is added or removed from the bioreactor during the course of cultivation. However in practice additions of air and acids or bases for pH control are made. Batch cultivation of suspension cells can be carried out in two types of bioreactors: Stirred tank and air lift reactors.

In airlift bioreactors for hybridoma cultivation, gas is introduced at the bottom of the vessel within the draught tube. A reduction in density of the aerated contents in the draught tube results in a circulation of the culture through the draught tube and down in the outer zone of the vessel. Advantages are that there are no moving parts or mechanical seals, there is adequate oxygen transfer, low hydrodynamic shear forces and low power input per unit volume. Operation: Batch operation with temperature controlled at 37 OC via a cooling jacket. pH is controlled by automatic addition of CO2 into the sparged gas or by NaOH. Dissolved oxygen tension is controlled by varying the concentration of oxygen in air. Foaming may be controlled by the addition of antifoam agent in conjunction with pluronic F-68. Scale Up: 10-2000L airlift bioreactors are used for cultivation of hybridoma cultures. Improved oxygen transfer rates have been shown at the larger scales. The increase in hydrostatic pressure resulting from increased bioreactor height does not have a deleterious effect on the cells. Media: Media supplement with 2-10 % animal sera as well as defined serum free media have been successfully used. Hybridoma cell lines of mouse, rat and human origin have been cultivated batch wise for 10 –17 days depending on the particular cell line. Hybridoma growth kinetics is not affected by dissolved oxygen tension in the range of 10 – 100 % saturation. Foaming is a problem at small scales and leads to the loss of cells and product