Biological reactors

Int J Artif Organs, 2002 Oct, 25(10), 1001 - 5
Primary human liver cells as source for modular extracorporeal liver support--a preliminary report; Sauer IM et al.; Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed . The use of porcine cells or tumor cell lines is controversial . Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation . The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient . A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion . A cell mass of 400 g - 600 g enables the clinical application of a liver lobe equivalent hybrid organ . A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous veno-venuous hemodiafiltration may be included if required . Cells from 54 human livers have been isolated (donor age: 56 +/- 13 years, liver weight: 1862 +/- 556 g resulting in a viability of 55.0 +/- 15.9%) . These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%) . Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure . The overall treatment time was 7-144 hours . No adverse events were observed . Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system.

Int J Artif Organs, 2002 Oct, 25(10), 994 - 1000
Cultivation of porcine hepatocytes in polyurethane nonwovens as part of a biohybrid liver support system; Linti C et al.; Many patients suffering from end-stage liver disease cannot be transplanted within reasonable time due to the shortage of donor organs . Bioartificial liver support systems may contribute to the liver regeneration or bridging the time until a liver graft for transplantation becomes available . Nonwovens with integrated oxygenation capacity have been developed and manufactured by melt blow technology using thermoplastic polyurethane . Capillary membranes for oxygenation were integrated into the nonwoven during the processing . The polyurethane nonwoven structures with adapted pore size and high pore volume allow high cell densities in the hepatocyte culture . The three-dimensional cell culture was housed by a flow bioreactor system and was integrated in a closed loop circulation with monitoring possibilities for pressure, pH, temperature, ammonia, and oxygen . Hepatocytes were isolated from rats or pigs by collagenase perfusion and infused into the medium-perfused circulation . Cells showed high viability and hepatocyte specific cytochrome P450-dependent metabolic function in culture (MEGX test).

Int J Artif Organs, 2002 Oct, 25(10), 975 - 84
A new bioassay including a small scale hepatocyte bioreactor for hepato-mediated toxicity testing in a target cell line; Deglmann CJ et al.; New approaches for in vitro testing of hepato-mediated toxicity are undertaken to offer alternatives to in vivo animal testing . The described bioassay for hepato-mediated toxicity testing is based on a small scale hepatocyte-bioreactor with pig hepatocytes connected to a silicon sensor based microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells and the microphysiometer alone . EAR represents the metabolic activity of tested cells (hepatocytes and ZR 751 cells) under the influence of perfused media, compared to controls, which were set to 100% . Cyclophosphamide (CYCL), whose cytostatic effect is dependent on CYP 450 biotransformation was used as a model substrate . CYCL showed decrease of EAR in hepatocytes, but not in ZR 751 cells . Bioreactor supernatant including CYCL was pumped into the microphysiometer and EARs of the target ZR 751 cell line were recorded . After 7 h of bioreactor supernatant perfusion the ZR 751 cell line showed an EAR decrease of 18.68% +/- 10.18, as compared to controls (bioreactor supernatant from the identical set-up without CYCL) . Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of CYCL by the bioreactor . This new bioassay serves as an example of future applications for hepatocyte bioreactors in automated toxicity testing devices, e.g . in preclinical drug studies or evaluation of hepato-mediated toxicity, not depending on cell destruction or further assays.

Int J Artif Organs, 2002 Oct, 25(10), 966 - 74
Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system; Abrahamse SL et al.; Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration . In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center . Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed . Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system . Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed . Subsequently, two groups were studied . In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours . In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control . After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated . The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production . However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.

Int J Artif Organs, 2002 Oct, 25(10), 960 - 5
ALEX (artificial liver for extracorporeal xenoassistance): a new bioreactor containing a porcine autologous biomatrix as hepatocyte support . Preliminary results in an ex vivo experimental model; Ambrosino G et al.; Long-term maintenance of viability and expression of differentiated hepatocyte function is crucial for bioartificial liver support . We developed a new bioreactor design (ALEX), associated with a new extracellular autologous hepatocyte biomatrix (Porcine Autologous Biomatrix - PBM) support . To test this new bioreactor, we compared it to a standard BAL (BioArtificial Liver) cartridge in a ex vivo model using human plasma added to bilirubin, ammonium and lidocaine . A pathology study was performed on both bioreactors . The results suggest that ALEX allows a maximal contact between the perfusing plasma and the liver cells and a proper hepatocyte support by a cell-to-matrix attachment . ALEX is a suitable cell support bioreactor, guaranteeing long-term maintenance of the metabolic activity of hepatocytes when compared to a standard BAL cartridge.

Int J Artif Organs, 2002 Oct, 25(10), 939 - 49
Advanced technology for extracorporeal liver support system devices; Borra M et al.; Acute Liver Failure (ALF) still presents high mortality rates, and liver transplant is the only treatment with proven efficacy . However transplant is not always possible and systems for Extracorporeal Liver Support (ELS) are being developed which can treat patients with ALF, for whom a transplant is not available, or is delayed . They can also treat patients with chronic liver disease who develop ALF . There are two types of ELS: artificial systems (hemoperfusion, plasmaperfusion, therapeutic plasma exchange, continuous hemodialysis and high volume continuous hemofiltration) and bioartificial systems . These are based on a biological component (animal or human hepatocytes) inserted into a bioreactor, whose main function is to perform the metabolic activity and synthesis that the liver can no longer perform . The results obtained in clinical trials have so far shown that the best results in terms of compensating for lost metabolic function and detoxification are obtained inserting artificial components in the bioartificial circuit.

ASAIO J, 2002 Nov-Dec, 48(6), 592 - 7
Development of a new bioartificial liver using a porcine autologous biomatrix as hepatocyte support; Ambrosino G et al.; Long-term maintenance of hepatocyte viability and differentiated function expression is crucial for bioartificial liver support . The maintenance of hepatocyte function in a bioreactor is still a problem . A major advance was the recognition that hepatocytes in attachment cultures can maintain their differentiation longer . To restore hepatocyte polarity and prolong their function, we developed a new bioreactor with a cross-flow geometry configuration and an original hepatocyte extracellular autologous biomatrix (Porcine Bio-Matrix) support . To test this new bioreactor, we compared it with a standard bioartificial liver cartridge in a suitable surgical model of acute liver failure in pigs . In our model, we performed a total hepatectomy, followed by partial liver transplantation after an 18 hour anhepatic phase . The results showed that the bioreactor containing the biomatrix was able to bridge the animal to transplantation and to sustain the transplanted liver until all function recovered (80% of animals survived, p = 0.0027) . No animal survived more than 24 hours after liver transplantation in the group treated with the traditional bioartificial liver, whereas hepatocyte viability on the Porcine Bio-Matrix was 65% after 12 hours of treatment . The results suggest that our biomatrix is a suitable cell support and guarantees long-term maintenance of metabolic activity of hepatocytes . Further studies are needed, but the results obtained with this new three-dimensional bioreactor are promising, and its potential is attractive.

Biorheology, 2003, 40(1-3), 331 - 6
Development and validation of a bioreactor for physical stimulation of engineered cartilage; Demarteau O et al.; A bioreactor has been developed to apply different regimes of physical stimulation to tissue specimens under highly controlled conditions . The computer-controlled device exposes specimens to compressive deformation at various strains and frequencies, measures the load applied to each sample and allows simultaneous medium stirring at different velocities . Validation tests confirmed the accuracy of the system in (i) its displacement (errors averaged 0.072+/-0.051 microm), and in (ii) setting the contact with the samples utilizing micrometer screws coupled to plungers (errors averaged 1.74+/-0.36% for samples of 1.60-3.18 mm thickness), thus ensuring accurate compressive deformation . The developed bioreactor, which represents an advance in the technology for physical stimulation of tissue specimens, is currently used to apply compressive deformation and hydrodynamic forces to human chondrocytes cultured in biodegradable polymer scaffolds, with the goals of (i) engineering functional grafts for the repair of cartilage defects (ii).

Biotechnol Bioeng, 2003 Jan 20, 81(2), 193 - 210
Enclosed outdoor photobioreactors: light regime, photosynthetic efficiency, scale-up, and future prospects; Janssen M et al.; Enclosed outdoor photobioreactors need to be developed and designed for large-scale production of phototrophic microorganisms . Both light regime and photosynthetic efficiency were analyzed in characteristic examples of state-of-the-art pilot-scale photobioreactors . In this study it is shown that productivity of photobioreactors is determined by the light regime inside the bioreactors . In addition to light regime, oxygen accumulation and shear stress limit productivity in certain designs . In short light-path systems, high efficiencies, 10% to 20% based on photosynthetic active radiation (PAR 400 to 700 nm), can be reached at high biomass concentrations (>5 kg {dry weight} m(-3)) . It is demonstrated, however, that these and other photobioreactor designs are poorly scalable (maximal unit size 0.1 to 10 m(3)), and/or not applicable for cultivation of monocultures . This is why a new photobioreactor design is proposed in which light capture is physically separated from photoautotrophic cultivation . This system can possibly be scaled to larger unit sizes, 10 to >100 m(3), and the reactor liquid as a whole is mixed and aerated . It is deduced that high photosynthetic efficiencies, 15% on a PAR-basis, can be achieved . Future designs from optical engineers should be used to collect, concentrate, and transport sunlight, followed by redistribution in a large-scale photobioreactor .

Biotechnol Bioeng, 2003 Jan 20, 81(2), 178 - 86
Characterization of gas-liquid mass transfer phenomena in microtiter plates; Hermann R et al.; Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method . The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system . The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters . To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter . The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells . The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors . The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material . Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed . This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area . This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks . In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L) . An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells .

Biodegradation, 2002, 13(2), 155 - 62
Biodegradation of VOCs from printing press air by an on-site pilot plant bioscrubber and laboratory scale continuous yeast cultures; Granstrom T et al.; The volatile organic compound composition (VOCs) of printing press air was found to contain mostly ethanol, but also ethyl acetate, 1-propanol, 2-propanol, 1-methoxy-2-propanol and 3-ethoxy-1-propanol . A pilot plant bioscrubber inoculated with a mixed microbial population was constructed on-site . The bioscrubber was able to treat the polluted gas efficiently . It, however, suffered from strong wall growth and blockages in the column . The efficiencies of the pilot plant and a bioreactor is compared . The yeasts Candida guilliermondii and Saccharomyces cerevisiae known to tolerate ethanol were selected instead of mixed population to avoid the wall growth a nd blockages inthe bioreactor . The removal of the VOCs both individually and as a complex mixture was tested in a microcultivation system and in continuous chemostat cultures with and without cell recycling . The Candida yeast could use all the compounds as a carbon source while growth of S . cerevisiae was markedly slower on the methoxylated and ethoxylated propanols . Best total removal of the VOCs was 99% and achieved by C . guilliermondii . The only compound that was not totally removable in the chemostat experiment with C . guilliermondii was 1-methoxy-2-propanol . In laboratory scale the total and volumetric removal of VOCs by C . guilliermondii was more efficient compared to the pilot plant encouraging to scale up and applying the yeast bioreactor to real field conditions.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 693 - 702
The anammox case-a new experimental manifesto for microbiological eco-physiology; Strous M et al.; The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater . It is also a new microbial physiology that was previously believed to be impossible . The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach . That approach is the focus of this paper . The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology . It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems . The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation . We argue that this was impossible using molecular ecology or conventional 'cultivation based techniques' alone . We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers.

Water Sci Technol, 2002, 46(9), 201 - 9
High salinity wastewater treatment using yeast and bacterial membrane bioreactors; Dan NP et al.; Two laboratory-scale membrane bioreactor systems were investigated to treat high salinity wastewater containing high organic (5,000 mg/L COD) and salt content (32 g/L NaCl), namely: (1) the Yeast Membrane Bioreactor (YMBR) and; (2) Yeast pretreatment followed by Bacterial Membrane Bioreactor (BMBR) . In the YMBR system, experimental runs were conducted with a mean biomass concentration of 12 g MLSS/L . Here the maximum COD removal rate of 0.93 g COD/g MLSS x day was obtained at F/M of 1.5 g COD/g MLSS.d . Whereas, the BMBR system was operated with a biomass concentration of up to 25 g MLSS/L, resulting in maximum COD removal rate of 0.32 kg COD/kg MLSS x day at F/M ratio of 0.4 . In comparison to BMBR, YMBR could obtain higher COD removal rate at higher organic loading, indicating the potential of a yeast reactor system to treat high salinity wastewater containing high organic concentration . Transmembrane pressure in BMBR was progressively increased from 2 to 60 kPa after 12 d, 6 d and 2 d at a hydraulic retention time (HRT) of 14 h, 9 h and 4 h, with average biomass concentration of 6.1, 15 and 20 g MLSS/L, respectively . Whereas the transmembrane pressure in YMBR has increased from 2 to 60 kPa only after 76 days of operation, with an average biomass concentration of 12 MLSS/L and an operating HRT range of 5-32 h.

Water Sci Technol, 2002, 46(9), 193 - 200
The effect of aeration and non-aeration time on simultaneous organic, nitrogen and phosphorus removal using an intermittent aeration membrane bioreactor; Ujang Z et al.; A laboratory-scale membrane bioreactor (MBR) was fed with synthetic wastewater to investigate the possibility of simultaneous removal of organic, nitrogen and phosphorus by intermittent aeration . The MBR consists of two compartments using a microfiltration membrane with 0.2 microm pore size and a surface area of 0.35 m2 . Hydraulic retention time was set at 24 hours and solid retention time 25 days . MLSS concentration in the reactor was in the range of 2,500-3,800 mg/L . The MLSS internal recycling ratio was maintained at 100% influent flow rate . Intermittent aeration was applied in this study to provide an aerobic-anaerobic cycle . Three stages of operations were conducted to investigate the effect of aeration and non-aeration on simultaneous organic and nutrient removal . In Stage 1, time cycles of aeration and non-aeration were set at 90/150 min and 150/90 min in the first and second compartment, the removal efficiency was 97%, 94% and 70% for COD, nitrogen and phosphorus respectively . In Stage 2, time cycles of aeration and non-aeration were set at 60/120 min and 120/60 min in the first and second compartment, the removal efficiency was 97%, 96% and 71% for COD, nitrogen and phosphorus respectively . In Stage 3, time cycles of aeration and non-aeration were set at 120/120 min and 120/120 min in compartment 1 and 2, the removal efficiency was 98%, 96% and 78% for COD, nitrogen and phosphorus respectively . Results show that longer non-aeration time in the second compartment provided better performances of biological phosphorus removal.

J Physiol, 2002 Nov 15, 545(Pt 1), 199 - 206
ATP release from human airway epithelial cells studied using a capillary cell culture system; Guyot A et al.; Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study . We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells . The Calu-3 cell line derived from human airway sub-mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a re-circulating medium pump . Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (approximately 250 microg (10(8) cells)(-1) day(-1)) . Elevating apical flow rate 5-fold increased ATP secretion from approximately 200 to 6618 fmol min(-1) . Reducing apical osmolarity by 25-43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained . Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis . Lowering apical {Ca(2+)} to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear-induced ATP release only in low-Ca(2+) solution, and therefore another secretory pathway may operate with physiological (i.e . mM) calcium . Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion.

J Microencapsul, 2002 Sep-Oct, 19(5), 571 - 90
Quantitative study of the production and properties of alginate/poly-L-lysine microcapsules; Gugerli R et al.; Alginate-polylysine-alginate (APA) microcapsules are of particular interest for their application as implants or for bioreactor cultures . Although their formation has been widely studied, there is still a lack of quantitative data describing resistance, membrane thickness and permeability . In this study, the quantitative application of a Texture Analyser for the measurement of capsule deformation yielded important results that permit comparison with other polymer systems used for encapsulation . Furthermore, single-membrane and multi-membrane capsules were formed in order to improve the modulation of the capsule properties . For single-membrane capsules, resistance was mostly affected by the incubation time in poly-L-lysine (PLL), the PLL molecular weight and concentration . The increase in resistance from 0.1 +/- 0.01 g/capsules to 2 +/- 0.2 g/capsules was linked to a membrane thickening (35-120 microm) and a decrease in permeability (150 to 40 kD) . Thus, it was not possible to modify resistance and membrane permeability independently . Multi-membrane capsules with a resistance comparable to single-membrane capsules could be formed using various combinations of PLL molecular weights, and enabled uncoupling of permeability and resistance properties.

Anal Chem, 2002 Nov 1, 74(21), 5507 - 12
Mass spectrometric immunoassay for parathyroid hormone-related protein; Lu CM et al.; This paper describes a novel two-site peptide immunoassay using the isotope 14C as the label and accelerator mass spectrometry as the detection system . A mouse monoclonal antibody (1A5) against the amino terminal region of human parathyroid hormone-related protein (PTHrP) was labeled with 14C by growing the hybridoma cells in a miniPERM bioreactor in the presence of {U-14C}L-leucine and {U-14C}D-glucose . The antibody was purified from the culture media using protein G affinity chromatography . The purified 14C-labeled antibody (14C-1A5) fractions showed excellent correlation between the levels of radioactivity and binding activity for PTHrP . Using 14C-1A5 as the detection antibody in a two-site immunoassay format for PTHrP1-141, a 16-kDa polypeptide, an analytic sensitivity of 10 pmol/L was achieved with a linear measurement range up to 1.3 nmol/L . Only approximately 17 pCi/ well (or 1.6 nCi/96-well microtiter plate) 14C-1A5 was used, which is far below the limit (50 nCi/g) for disposal as nonradioactive waste . This study may serve as a model for the development of sensitive and "nonradioactive" immunoassays for peptides, including polypeptide tumor markers.

Biotechnol Bioeng, 2003 Jan 5, 81(1), 106 - 14
The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage; Saarinen MA et al.; The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture . Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP) . Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation . Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored . The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively . DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line . In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration . Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells . TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration . It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells .

Biotechnol Bioeng, 2003 Jan 5, 81(1), 80 - 91
Unveiling steady-state multiplicity in hybridoma cultures: the cybernetic approach; Namjoshi AA et al.; Mammalian cells grown in suspension produce waste metabolites such as lactate, alanine, and ammonia, which reduce the yield of cell mass and the desired product on the nutrients supplied . Previous studies (Cruz et al., 1999; Europa et al., 2000; Follstad et al., 1999) have shown that the cells can be made to alter their metabolism by starving them on their nutrients in continuous cultures at low dilution rates or starting the culture as a fed-batch . This leads to multiple steady states in continuous reactors, with some states being more favorable than others . Mathematical models that take into account the metabolic regulation that leads to these multiple steady states are invaluable tools for bioreactor control . In this article we present a cybernetic modeling strategy in which Metabolic Flux Analysis (MFA) is used to guide the cybernetic formulation . The hybridoma model presented as a result of this strategy considers the partially substitutable, partially complementary nature of glucose and glutamine . The choice of competitions within the network is guided by MFA and the model is successful in explaining the three multiple steady states observed . The cybernetic model though identified for the hybridoma experiments of Hu and others (Europa et al., 2000) seem generally applicable to mammalian systems as it captures the pathways that are common to mammalian cells grown in suspension . The model presented here could be used for start-up strategies for continuous reactors and model-based feedback control for maintaining high productivity of the reactor .

Biotechnol Bioeng, 2003 Jan 5, 81(1), 66 - 73
Distributed model of solid waste anaerobic digestion: effects of leachate recirculation and pH adjustment; Vavilin VA et al.; A distributed model of solid waste digestion in a 1-D bioreactor with leachate recirculation and pH adjustment was developed to analyze the balance between the rates of polymer hydrolysis/acidogenesis and methanogenesis during the anaerobic digestion of municipal solid waste (MSW) . The model was calibrated on previously published experimental data generated in 2-L reactors filled with shredded refuse and operated with leachate recirculation and neutralization . Based on model simulations, both waste degradation and methane production were stimulated when inhibition was prevented rapidly from the start, throughout the reactor volume, by leachate recirculation and neutralization . An optimal strategy to reduce the time needed for solid waste digestion is discussed .

J Biomed Sci, 2002 Nov-Dec, 9(6 Pt 2), 631 - 8
Establishment of a human somatic hybrid cell line for recombinant protein production; Cho MS et al.; Cell fusion techniques were used to derive mammalian host cell lines suitable for large-scale production of therapeutic proteins . Although the 293S cell line, of human embryonic kidney origin, is an excellent host cell for mammalian gene expression, these cells have a tendency to form large and tight aggregates in suspension cultures and bioreactors . To solve the problem of aggregation, 293S cells were fused to a human suspension cell line, 2B8 (a Burkitt's lymphoma derivative), using polyethylene glycol (PEG) . The PEG-treated 293S and 2B8 cells were selected in a medium supplemented with hypoxanthine-aminopterin-thymidine and G418 (1 mg/ml) to eliminate nonfused cells . These hybrid clones, designated as HKB (hybrid of kidney and B cells), are negative for endogenous immunoglobulin expression . Most clones are readily adaptable to serum-free suspension culture under shaking conditions without forming large and tight aggregates . One clone, HKB11, was shown to support high-level expression of cytokines {interleukin (IL)-2 and IL-4}, ICAM-1 and rFVIII in a side-by-side comparison with 293 and Chinese hamster ovary cells . The above-described characteristics of HKB cells indicate that HKB11 is a favorable cell host for the production of human therapeutic proteins .

Cells Tissues Organs, 2002, 172(2), 96 - 104
Skin genetically engineered as a bioreactor or a 'metabolic sink'; Christensen R et al.; Genetically manipulated human keratinocytes can produce and secrete medically relevant proteins to the circulation . Genetically modified skin may also function as a 'metabolic sink' detoxifying the body of metabolites which accumulate in certain metabolic diseases . At the National Institutes of Health (NIH), Bethesda, Md., a clinical trial investigating the treatment of an ocular disease using the skin as a 'metabolic sink' for ornithine accumulating in gyrate atrophy patients is being prepared . The trial will involve the transplantation of a small patch of autologous keratinocytes, transduced ex vivo, onto the thighs of patients with gyrate atrophy . We are now investigating other diseases where this technology may be applicable such as in the treatment of hyperphenylalaninemia or hypercholesterolemia .

J Biotechnol, 2003 Jan 23, 100(2), 141 - 6
Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor; Osinga R et al.; Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum . The food was supplied either as intact algae or as a filtered crude extract . Growth (measured as an increase in underwater weight) was found in both experiments . The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days . The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days . These results show that it is possible to grow a sponge using a single microorganism species as a food source . In addition, it was demonstrated that sponges are also capable of growing on non-particulate food . Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures.

Folia Microbiol (Praha), 2002, 47(4), 417 - 21
Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings . Application to the decolorization of synthetic dyes; Rodriguez Couto S et al.; The production of manganese-dependent peroxidase (MnP) by Phanerochaete chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated . Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 mukat/L were obtained, respectively . The in vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed . The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%).

Water Res, 2002 Nov, 36(18), 4605 - 15
Predicting oxygen transfer and water flow rate in airlift aerators; Burris VL et al.; Water flow rate, gas-phase holdup, and dissolved oxygen (DO) profiles are measured in a full-scale airlift aerator as a function of applied air flow rate . A model that predicts oxygen transfer based on discrete-bubble principles is applied . The riser DO profiles are used to calculate the initial bubble size . The range of calculated bubble diameters obtained using the model is 2.3-3.1 mm . The Sauter-mean diameter of bubbles measured in the laboratory ranged from 2.7 to 3.9 mm . The riser and downcomer DO profiles and gas holdups predicted by the model are in close agreement with the experimental results . A model that predicts water flow rate based on an energy balance is used to calculate Kt, the frictional loss coefficient for the air-water separator . Excluding the data at the very lowest air flow rate, the range of calculated values for Kt (3-8) is close to a literature value of 5.5 proposed for hydrodynamically similar external airlift bioreactors . The models should prove useful in the design and optimization of airlift aerators.

Water Res, 2002 Nov, 36(18), 4445 - 54
Enzyme production activity of Phanerochaete chrysosporium and degradation of pentachlorophenol in a bioreactor; Shim SS et al.; Lignin peroxidase production by a white rot fungus, Phanerochaete chrysosporium, was experimentally investigated using a batch system and a reactor system with various carriers . Immobilization of mycelia cell culture was more effective in promoting cell growth and lignin peroxidase production compared to conventional stationary liquid culture . Biostage carrier, commonly used for biochemical treatment in a fluidized bed disposal system, greatly improved production of lignin peroxidase up to 8.1 U/mL in the batch system . The packed bed reactor system was operated using a repeated batch technique, consisting of alternating growth and production phases, to sustain lignin peroxidase growth and production during the entire experiment period . Steady-state continuous PCP degradation over an extended period was accomplished with a mineralization ratio exceeding 80% . These systems and operation methods are promising techniques for the treatment of hazardous waste.

J Biotechnol, 2003 Jan 9, 100(1), 13 - 22
Plant-cell bioreactors with simultaneous electropermeabilization and electrophoresis; Yang RY et al.; Experimental investigations on using low-level electric currents and voltages to extract, transport, and collect intracellular secondary metabolites from plant cells while maintaining their viabilities were conducted focusing on the production of: (1) ionic betalains, mainly negatively-charged betanin, from Beta vulgaris cells, and (2) ionic alkaloids, particularly positively-charged ajmalicine and yohimbine, from Catharanthus roseus cells . Three versions of tubular membrane reactors in which electropermeabilization of cell membranes and electrophoresis and diffusion of ionic products take place simultaneously, with or without convective flow, to achieve desirable extraction were developed . Concentrations of secondary metabolites produced from these plant-cell reactors under steady and oscillatory electrical forcings were recorded and the viabilities of treated cells examined . Oscillatory application of electrical field appears to produce more products while retaining higher cell viability.

J Ind Microbiol Biotechnol, 2002 Nov, 29(5), 268 - 74
Effect of nickel deprivation on methanol degradation in a methanogenic granular sludge bioreactor; Zandvoort MH et al.; The effect of omitting nickel from the influent on methanol conversion in an Upflow Anaerobic Sludge Bed (UASB) reactor was investigated . The UASB reactor (30 degrees C, pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT) and at organic loading rates (OLRs) ranging from 2.6 to 7.8 g COD l reactor(-1) day(-1) . The nickel content of the sludge decreased by 66% during the 261-day reactor run because of washout and doubling of the sludge bed volume . Nickel deprivation initially had a strong impact on the methanogenic activity of the sludge with methanol; e.g., after 89 days of operation, this activity was doubled by adding 2 micro M nickel . Upon prolonged UASB reactor operation, methanol and VFA effluent concentrations decreased whereas the sludge lost its response to nickel addition in activity tests . This suggests that a less nickel-dependent methanol-converting sludge had developed in the UASB reactor.

J Ind Microbiol Biotechnol, 2002 Nov, 29(5), 259 - 63
Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures; Papagianni M et al.; The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions . Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads . Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor . Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus . Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g(-1), while glucoamylase specific activity increased from 205 to 350 U g(-1) . The effect was more pronounced in bioreactor cultures . A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor.

Appl Environ Microbiol, 2002 Nov, 68(11), 5367 - 73
RNA stable isotope probing, a novel means of linking microbial community function to phylogeny; Manefield M et al.; Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology . Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it . In this study stable-isotope-labeled {13C}phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process . Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis . In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus . Our results suggest that this organism is important for the function of this bioreactor.

Altern Lab Anim, 2002 Sep-Oct, 30(5), 525 - 38
Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions; Zeilinger K et al.; In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry . A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation . We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells . The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation . In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (CYP450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture . Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value . In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture . After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver . Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture . Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor . This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.

Altern Lab Anim, 2002 Sep-Oct, 30(5), 515 - 23
Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor, a static-cell bioreactor and static cell cultures; Pazos P et al.; In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects . The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse, Integra, Wallisellen, Switzerland) and a static cell bioreactor system (CELLine CL 6-well, Integra), and to compare them with the use of conventional cell culture flasks . A human cell line, Int 407, was exposed to cadmium chloride (CdCl(2); 10-(7-)10-(8)M) for 4 weeks . Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay . In addition, total protein determination and cadmium uptake measurements were performed . The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl(2) concentration . However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl(2) . Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl(2) concentration applied and the system used . In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies . It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.

Biometals, 2002 Dec, 15(4), 377 - 90
Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review; Mallick N; This presentation comprises a review on the use of immobilized algae for wastewater nitrogen, phosphorus and metal removal purposes . Details of the use of immobilized algae, the techniques of immobilization and the effects of immobilization on cell function are included . Particularly relevant in their use for heavy metal removal from wastewaters; upon enriching the biomass in metal, can be recoverd, thereby providing economic advantages . The use of immobilized microalgae in these processes is very adequate and offers significant advantages in bioreactors . The future of this area of algal cell biotechnology is considered.

Water Res, 2002 Sep, 36(16), 3941 - 8
A tanks-in-series bioreactor to simulate macromolecule-laden wastewater pretreatment under sewer conditions by Aspergillus niger; Coulibaly L et al.; Sewers are typically a means of transporting wastewater to a treatment facility, with little biotransformation of the soluble polymeric organic matter by suspended biomass . In the interest of providing an effective pretreatment of wastewater in a sewer network, it is necessary to design an accurate tool simulating sewer conditions and introduce an appropriate biomass for macromolecular pollutant degradation . Such a model reactor was built using a tanks-in-series design and the degradation of a polysaccharide (starch) by Aspergillus niger MUCL 28817 was studied . Starch degradation and the accumulation of intermediates (hydrolysis fragments) in the individual reactors were quantified under transient conditions, at a mean hydraulic residence time of 17 h . Starch was degraded by 90% in this reactor system and an accumulation of oligosaccharides with molecular weight lower than 1,000 Da was observed . These results may be helpful in the development of wastewater treatment in sewers and in the alleviation of the burden on undersized wastewater treatment systems.

Biotechnol Bioeng, 2002 Dec 30, 80(7), 794 - 805
Modeling, optimization and experimental assessment of continuous L-(-)-carnitine production by Escherichia coli cultures; Alvarez-Vasquez F et al.; In a previous paper Canovas et al . (Biotechnol Bioeng 2002;77:764-775) presented a model for L-(-)-carnitine production using Escherichia coli O44 K74, in a cell-recycle bioreactor for the biotransformation of crotonobetaine into L-carnitine . In this work we optimize this biotechnological setup and experimentally verify the predicted optimal parameter profiles . Provided with a reliable and robust S-system description of the cell-bioreactor combined system, we applied the Indirect Optimization Method described by Torres et al . (Biotechnol Bioeng 1997;55(5):758-772; Food Technol Biotechnol 1998;36(3):177-184) . This optimization approach provides different parameter value profiles, all of which are compatible with the cell physiology and the bioreactor operating conditions, that yield increased rates of L-(-)-carnitine production . Three parameters were seen to be of critical importance for maximizing L-(-)-carnitine production: the dilution rate, the initial crotonobetaine concentration, and the carnitine dehydratase activity . When the first two were changed in the experimental setup, there was a 74% increase in the L-(-)-carnitine production rate, performance that was in close agreement with the predictions of the model . In accordance with the optimized solution, a further improvement (90% increase in the L-(-)-carnitine production rate) could be attained by over-expressing up to 5 times the carnitine dehydratase basal activity . Thus the optimization approach shown herein provides experimental evidence of a new strategy which demonstrates the possible variables that can be subjected to modifications compatible with the cell physiology and bioreactor operating conditions, and which are able to yield increased rates of L-(-)-carnitine production .

Biotechnol Bioeng, 2002 Dec 30, 80(7), 746 - 54
Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains; Gerigk M et al.; A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E . coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine . Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype . With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities . With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided . The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors .

Biotechnol Bioeng, 2002 Dec 30, 80(7), 719 - 30
Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells; Lengwehasatit I et al.; Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells . This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis . During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells . This event was paralleled by a simultaneous increase in GADD153 expression . The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented . In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress . However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment . Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death . These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment .

Curr Opin Crit Care, 2002 Apr, 8(2), 171 - 7
Extracorporeal support of the failing liver; Mitzner SR et al.; Successes in machine-based extracorporeal support for different organ functions stimulated research in the field of liver support approximately 50 years ago . Initial failure to improve outcome using detoxification methods like dialysis, blood and plasma exchange, or plasmapheresis over sorbents fueled interest in biologic liver support concepts using bioreactors or combined methods . New device configurations, technical improvement of existing detoxification methods, and the refinement in cell culture techniques led to a boost in research on biologic and nonbiologic approaches . Currently, many systems are in the preclinical phase or have entered clinical studies . A number of completed clinical trials have reported a favorable therapeutic impact of the most advanced solutions on the course and outcome of liver failure . Often, findings must be reconfirmed . However, current knowledge suggests that extracorporeal liver support can successfully stabilize liver function, improve the clinical condition of patients, and considerably improve survival in certain subgroups of patients with fulminant hepatic failure and acute decompensation of chronic hepatic failure . Although the initial focus of liver support methods was bridging to liver transplantation, bridging to recovery of organ function and treatment of intractable pruritus are now valuable indications.

J Biotechnol, 2002 Nov 13, 99(3), 249 - 57
Enzymatic membrane reactors for biodegradation of recalcitrant compounds . Application to dye decolourisation; Lopez C et al.; Membrane bioreactors are being increasingly used in enzymatic catalysed transformations . However, the application of enzymatic-based treatment systems in the environmental field is rather unusual . The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation . Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment . Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems . Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented.

J Biotechnol, 2002 Nov 13, 99(3), 237 - 48
Sensor fusion for on-line monitoring of yoghurt fermentation; Cimander C et al.; Measurement data from an electronic nose (EN), a near-infrared spectrometer (NIRS) and standard bioreactor probes were used to follow the course of lab-scale yoghurt fermentation . The sensor signals were fused using a cascade neural network: a primary network predicted quantitative process variables, including lactose, galactose and lactate; a secondary network predicted a qualitative process state variable describing critical process phases, such as the onset of coagulation or the harvest time . Although the accuracy of the neural network prediction was acceptable and comparable with the off-line reference assay, its stability and performance were significantly improved by correction of faulty data . The results demonstrate that on-line sensor fusion with the chosen analyzers improves monitoring and quality control of yoghurt fermentation with implications to other fermentation processes.

Water Sci Technol, 2002, 46(6-7), 199 - 206
Non-point source pesticide removal by a mountainous wetland; Kao CM et al.; Non-point source (NPS) pollution is believed to be one of the major causes of impairment of water bodies . Among NPS pollution, agricultural NPS pollution is considered to be the largest single category resulting in water quality deterioration . Pesticides are some the most ubiquitous of these agricultural NPS pollutants . In this study, a mountainous wetland was selected to investigate the effects of the natural wetland system on the NPS pesticide (atrazine) removal to maintain the surface water quality . The selected wetland receives water from two unnamed creeks, which drain primarily upgradient agricultural lands . Wetland investigation and monitoring were conducted from November 1999 to March 2001 . Major storm events and baseline water quality samples were analyzed . Field results indicate that the wetland was able to remove NPS atrazine flushed from the upgradient agricultural lands after the occurrence of storm events . Laboratory aerobic and anaerobic bioreactor experiments were conducted to evaluate the biodegradation of atrazine under the intrinsic conditions of the wetland system . Microbial enumeration was conducted for a quick screen of bacterial activity in the studied wetland . Results from the study suggest that the methanogenesis process was possibly the dominant biodegradation pattern, and atrazine can be degraded under reductive dechlorinating conditions when sufficient intrinsic organic matter was provided . Results from this study can provide us with further knowledge on pesticide removal mechanisms in natural wetlands and evaluate the role of wetlands in controlling pesticide pollutants from stormwater runoff.

Biotechnol Bioeng, 2002 Dec 20, 80(6), 677 - 84
Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence-based digital image analysis; Hassan M et al.; Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus . After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen . The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera . Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells . Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis . At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability . The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area) . This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor . Estimated viable biomass during fermentation was strongly influenced by the process conditions . The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T . harzianum .

Biotechnol Bioeng, 2002 Dec 20, 80(6), 632 - 6
New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale; Buziol S et al.; Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks . As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation . The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor . For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed . A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber . Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified . The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli .

Huan Jing Ke Xue, 2002 Jul, 23(4), 67 - 70
{Studies on ceramic dual function membrane bioreactor for wastewater treatment}; Zhang Y et al.; A new bioreactor including ceramic dual function membrane, filtration and aeration, was developed and used for wastewater treatment . The turbidity of effluent by the ceramic membrane was comparable to that of 4-24 hours by sedimentation . Aeration and filtration could be switched constantly to the ceramic membrane in the bioreactor and the clogging of ceramic membrane could be well prevented . The wastewater was treated in batch and continues operation modes respectively and the experimental results showed that the COD loading was 1.1 kg/(m3.d) and 1.5 kg/(m3.d) respectively.

Huan Jing Ke Xue, 2002 Jul, 23(4), 42 - 6
{Treatment of a 2,4-dichlorophenol contaminated wastewater in an air-lift inner-loop bioreactor}; Quan X et al.; An air lift inner-loop bioreactor packed with honeycomb-like ceramic carrier was immobilized with a 2,4-Dichlorophenol-degrading pure culture and was investigated to degrade 2,4-Dichlorophenol and phenol . In fed-batch operation mode, 2,4-DCP biodegradation rate increased with run numbers and followed zero-order kinetics model when it existed alone, but when 2,4-DCP was present in the mixture with phenol, phenol degradation rate had an apparent trend to increase whereas 2,4-DCP removal rate became slower and slower . In continuous operation, 2,4-DCP at the concentration ranged from 6.9 to 102.4 mg/L could be degraded well at the dilution rate of 0.16 h-1 and the average removal percentage was 96.5% . Carbon sources changed from 2,4-DCP to acetate sodium and peptone in the course of operation for 12 days did not cause the bacteria loss the DCP-degrading ability.

Exp Dermatol, 2002 Oct, 11(5), 456 - 61
Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin; Meng X et al.; Using the plasmid DNA injection method, we introduced cytokine genes into skin to determine whether systemic expression of cytokine genes is possible . Eight human cytokine {interleukin-4 (IL-4), IL-6, IL-10, transforming growth factor beta1 (TGF-beta1), monocyte chemotactic and activating factor (MCAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)} gene expression vectors were constructed and injected into rat skin . Transgenic cytokines in local keratinocytes and in the sera were assayed with ELISA . Our results showed that transgenic cytokines were markedly increased in keratinocytes at the injection site . The serum concentrations of IL-4, 6, 10 and TauGF-beta1 reached levels high enough to have systemic biologic effects . However, other cytokines used in this study could not be detected in the sera . Moreover, the serum transgenic IL-10 level after subcutaneous injection was significantly higher than after intramuscular injection . We suggest that keratinocytes can be used as a bioreactor to achieve systemic expression of cytokine genes by DNA injection, but the transgenic protein level in circulation depends on different kinds of cytokine . This level also depends on different target cells used for gene transfer.

J Gravit Physiol, 2001 Dec, 8(2), 57 - 66
Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat; Bhat GK et al.; The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system . The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells . A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3) . These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions . It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls . Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels . Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV . Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h . Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h . It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone . Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

Gene Ther, 2002 Oct, 9(20), 1351 - 8
Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation; Nicol F et al.; Intramuscular (i.m.) injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels . To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m . injection and electroporation . With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two- to eight-fold higher levels of SEAP in mouse serum than plasmid in saline . Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15-50 kDa poly-L-glutamate consistently yielded the highest expression levels . The poly-L-glutamate formulation was effective in two different muscle groups in mice at various plasmid doses for several transgenes, including an erythropoietin (EPO) gene, for which expression was elevated four- to 12-fold in comparison to animals that received EPO plasmid in saline . Transgene expression was localized to myofibers . Poly-L-glutamate may improve transgene expression in part by increasing plasmid retention in skeletal muscle . Poly-L-glutamate did not enhance gene transfer in the absence of electroporation . Therefore, the polymer is a novel formulation that specifically enhances the transfer and expression of genes delivered with electroporation.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 1003 - 9
Continuous selective extraction of secondary metabolites from Catharanthus roseus hairy roots with silicon oil in a two-liquid-phase bioreactor; Tikhomiroff C et al.; A two-liquid-phase bioreactor was designed to extract indole alkaloids from Catharanthus roseus hairy roots with silicon oil . Partition studies between silicon oil and culture medium showed that the silicon oil did not alter the availability of nutrients . The affinity of tabersonine and lochnericine for silicon oil is nine times higher than for the aqueous phase . Cultures were elicited with 25 mg/L of jasmonic acid . The growth of the hairy roots was not significantly modified by the presence of silicon oil . The overall specific yields of tabersonine and lochnericine were increased by 100-400% and 14-200%, respectively, with the use of silicon oil in nonelicited control cultures . In elicited cultures, these values were 10-55% for tabersonine and 20-65% for lochnericine . Serpentine was never found in the silicon oil . All measured alkaloids' specific yields were higher using silicon oil and elicitation, suggesting that the silicon oil, while acting as a metabolic sink for tabersonine and lochnericine, was efficient in increasing metabolic fluxes of the secondary metabolism pathways.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 975 - 85
Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions; Travascio P et al.; The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated . Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports . Butyl methacrylate (BMA) was used as graft monomer . Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme . Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields . By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent . Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor . Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained . The addition of methanol reduced the non-isothermal activity increase . The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 964 - 8
Polymerization of glucans by enzymatically active membranes; Becker M et al.; Conventional enzyme membrane reactors are not appropriate for a continuous synthesis of macromolecules and simultaneous product release . By immobilizing the enzyme in sufficiently large pores of a membrane an ensemble of miniaturized bioreactors is created . Product molecules are continuously removed from the enzyme by the flow of the reaction mixture across the membrane . Additionally, by varying the flow rate, it ought to be possible to influence the substrate as well as the enzyme-product residence times and thereby the product macromolecule's size . In this paper we present the first results of experiments involving enzymatic 1,4-alpha-glucan synthesis, using sucrose as substrate, maltooligosaccharides (DP 3-6) as primers, and membrane-immobilized amylosucrase . Epoxy groups for a covalent enzyme immobilization were generated on polypropylene microfiltration membranes by heterogeneous photoinitiated graft polymerization of glycidyl methacrylate . The influence of primer concentration and flow rate through the enzyme-membrane on amylosucrase activity, molecule growth, and coupling efficiency for glucose (% of coupled glucose versus free glucose) were investigated . The enzymatically mediated chain elongation of maltooligosaccharides by the successive addition of glucose units was achieved for the first time in a transmembrane process utilizing amylosucrase membranes.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 951 - 63
Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering; Williams KA et al.; Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs . Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor . The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor . Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth . Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform . Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation) . These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth . Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.

Acta Biochim Pol, 2002, 49(2), 313 - 21
Antiangiogenic gene therapy in inhibition of metastasis; Szala S et al.; This short review attempts to demonstrate the usefulness of antiangiogenic gene therapy in achieving inhibition of growth in experimentally-induced metastases . Certain normal tissues (for example skeletal muscle) may be used in vivo, after genetic modification, as a "bioreactor", able to produce and secrete into the bloodstream proteins known to exert antiangiogenic effects . By inhibiting neoangiogenesis these proteins would thus prevent the development of metastases . The review discusses also the perspectives of antimetastatic therapy based on certain types of allogenic cells (for example myoblasts and fibroblasts) that had been genetically modified and then microencapsulated . The strategy of encapsulation is aimed at protecting the modified cells secreting antiangiogenic factors from being eliminated by the immune system . Secretion of antiangiogenic proteins by these microencapsulated cells can be controlled with inducible promoters . Antiangiogenic genes remaining under the transcriptional control of such promoters may be switched on and off using antibiotics, such as tetracycline derivatives, or steroid hormones.

Nutrition, 2002 Oct, 18(10), 842 - 8
Insulin secretion and sensitivity in space flight: diabetogenic effects; Tobin BW et al.; Nearly three decades of space flight research have suggested that there are subclinical diabetogenic changes that occur in microgravity . Alterations in insulin secretion, insulin sensitivity, glucose tolerance, and metabolism of protein and amino acids support the hypothesis that insulin plays an essential role in the maintenance of muscle mass in extended-duration space flight . Experiments in flight and after flight and ground-based bedrest studies have associated microgravity and its experimental paradigms with manifestations similar to those of diabetes, physical inactivity, and aging . We propose that these manifestations are characterized best by an etiology that falls into the clinical category of "other" causes of diabetes, including, but not restricted to, genetic beta-cell defects, insulin action defects, diseases of the endocrine pancreas, endocrinopathies, drug or chemically induced diabetes, infections, immune-mediated metabolic alteration, and a host of genetic related diseases . We present data showing alterations in tumor necrosis factor-alpha production, insulin secretion, and amino acid metabolism in pancreatic islets of Langerhans cultured in a ground-based cell culture bioreactor that mimics some of the effects of microgravity . Taken together, space flight research, ground-based studies, and bioreactor studies of pancreatic islets of Langerhans support the hypothesis that the pancreas is unable to overcome peripheral insulin resistance and amino acid dysregulation during space flight . We propose that measures of insulin secretion and insulin action will be necessary to design effective countermeasures against muscle loss, and we advance the "disposition index" as an essential model to be used in the clinical management of space flight-induced muscle loss.

Biomaterials, 2002 Dec, 23(24), 4793 - 801
Cardiac tissue engineering: characteristics of in unison contracting two- and three-dimensional neonatal rat ventricle cell (co)-cultures; van Luyn MJ et al.; Patients with heart failure have, in spite of improved palliative therapies, bad prognosis . Cardiac tissue engineering by use of a temporary bioscaffold and cardiomyocytes may help to find answers for future treatments in heart failure . For that purpose two neonatal rat heart ventricular cell fractions were obtained after a gradient cell separation . Time related characteristics of Fractions I and II were established in two-dimensional (2-D) and three-dimensional (3-D) cell cultures . The 3-D cardiac constructs were obtained by use of a bovine type I collagen matrix after culturing either under static conditions or in the HARV bioreactor . With the 2-D cultures contracting cells were present after 1 day, and reached confluency from day 5 on and this was maintained up to 135 days . In Fraction-I some non-contracting cells were always noticed between the (in time in unison) contracting cells . Transmission electron microscopy (TEM) revealed that these mainly concerned fibroblasts . Differences in the expression of alpha-SM-1 actin and troponin-T were observed between the two fractions . In both fractions endothelial cells and macrophages were only sporadically observed . All through the 3-D matrix pendant-like single cell and clustered cell contractions were present after 1-2 days, resulting in time in unison contracting of cells with the collagen matrices . The whole event was faster with Fraction-I and was observed up to 3 weeks . At this time point clusters of troponin-T positive cells were found scattered through the collagen matrices . Additionally, TEM revealed healthy layers of connected cardiomyocytes with intercalated discs, in this case on and in between the collagen fibres . These findings provide evidence that in unison contracting structurally organized cell-matrix cardiac constructs can be engineered by use of co-cultures (neonatal cardiomyocytes and fibroblasts) and collagen matrices, which is very promising for the repair of larger scar areas of the myocardium .

Water Sci Technol, 2002, 46(4-5), 67 - 76
Pilot-scale testing membrane bioreactor for wastewater reclamation in industrial laundry; Andersen M et al.; A pilot-scale study of membrane bioreactor treatment for reclamation of wastewater from Berendsen Textile Service industrial laundry in Soborg, Denmark was carried out over a 4 month period . A satisfactory COD degradation was performed resulting in a low COD in the permeate (< 50 mg/l) . To obtain satisfactory treatment, addition of nitrogen was necessary . The biodegradability of the permeate was very low (BOD5 < 2 mg/l) . A hydraulic retention time of 1 d turned out to be sufficient at a sludge concentration of 10 g MLSS/l . Through addition of a cationic polymer, a satisfactory dewaterability of the sludge was reached . Membrane tests showed that operating at a trans-membrane pressure of 3 bar and a cross-flow velocity of 4 m/s, a flux of 120 l/m2h can be expected without using chemicals for membrane cleaning . The quality of the permeate was very good when comparing to the reuse quality demands of water to the wash processes . Reuse of the permeate in all rinsing steps requires additional treatment through reverse osmosis.

Water Sci Technol, 2002, 46(4-5), 281 - 6
Hanced biological phosphorus removal in membrane bioreactors; Adam C et al.; Enhanced biological phosphorus removal (Bio-P) in a membrane bioreactor (MBR) promises several advantages but was never attempted as not compatible with high sludge ages . This article includes description and results of bench-scale investigations on Bio-P removal in an MBR . An MBR bench-scale plant (210 L) was operated in parallel to a conventional WWTP under comparable process conditions . The results show that Bio-P removal is possible in MBR . The effluent qualities of the plants were comparable . The effluent P-concentration was always lower than 0.2 mg P(T)/L . In the MBR bench-scale plant P-uptake occurred mainly in the anoxic zone . Investigations with P-spiking showed higher Bio-P potential as P-removal increased up to 20-25 mg/L while P/TS rose up to >6%.

Water Sci Technol, 2002, 46(4-5), 19 - 26
Coarse media filtration for enhanced primary treatment of municipal wastewater; Liao Z et al.; In these experimental studies enhanced primary treatment in coarse media filters has been evaluated . The coarse media used in the filters have been plastic carriers normally used for moving bed bioreactors, a Kaldnes carrier K1 with density < 1 g/cm3 (K1L) and a bigger carrier K2, that was used both in a lighter, floating (K2L) version and a heavier, sinking one (K2H) . The results demonstrated that media characteristics significantly influenced filter performance . The dosing of a high MW and high charge cationic polymer was, however, found to have an even greater effect on treatment efficiency and especially on head loss development . Both filters were found to be feasible for enhanced primary treatment at high filtration rates (20-30 m/h) especially when dosing low dosages (1-3 mg/l) of polymer . The combination of two media into a dual media filter might take the advantages of both . Further experiments were carried out on a dual media filter consisting of lighter K1L media and heavier K2H media . It was indeed found that the K1L+K2H up-flow filter resulted in better performances in terms of water production when an optimised backwash procedure was taken into account.

Biotechnol Bioeng, 2002 Dec 5, 80(5), 569 - 79
Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions; Xie L et al.; PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions . Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8 . The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates . Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions . It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced . Adenovirus infection resulted in profound changes in cell growth and metabolism . Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection . Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH . The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study . Deviations from this optimum resulted in significant reductions of virus productivity . The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production .

Circulation, 2002 Sep 24, 106(12 Suppl 1), I143 - 50
Tissue engineering of functional trileaflet heart valves from human marrow stromal cells; Hoerstrup SP et al.; BACKGROUND: We previously demonstrated the successful tissue engineering and implantation of functioning autologous heart valves based on vascular-derived cells . Human marrow stromal cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically . The feasibility of creating tissue engineered heart valves (TEHV) from MSC as an alternative cell source, and the impact of a biomimetic in vitro environment on tissue differentiation was investigated . METHODS AND RESULTS: Human MSC were isolated, expanded in culture, and characterized by flow-cytometry and immunohistochemistry . Trileaflet heart valves fabricated from rapidly bioabsorbable polymers were seeded with MSC and grown in vitro in a pulsatile-flow-bioreactor . Morphological characterization included histology and electron microscopy (EM) . Extracellular matrix (ECM)-formation was analyzed by immunohistochemistry, ECM protein content (collagen, glycosaminoglycan) and cell proliferation (DNA) were biochemically quantified . Biomechanical evaluation was performed using Instron(TM) . In all valves synchronous opening and closing was observed in the bioreactor . Flow-cytometry of MSC pre-seeding was positive for ASMA, vimentin, negative for CD 31, LDL, CD 14 . Histology of the TEHV-leaflets demonstrated viable tissue and ECM formation . EM demonstrated cell elements typical of viable, secretionally active myofibroblasts (actin/myosin filaments, collagen fibrils, elastin) and confluent, homogenous tissue surfaces . Collagen types I, III, ASMA, and vimentin were detected in the TEHV-leaflets . Mechanical properties of the TEHV-leaflets were comparable to native tissue . CONCLUSION: Generation of functional TEHV from human MSC was feasible utilizing a biomimetic in vitro environment . The neo-tissue showed morphological features and mechanical properties of human native-heart-valve tissue . The human MSC demonstrated characteristics of myofibroblast differentiation.

J Urol, 2002 Oct, 168(4 Pt 2), 1794 - 7
Engineering of human cartilage rods: potential application for penile prostheses; Kim BS et al.; PURPOSE: Natural penile prostheses created from the patient's own cells may eliminate the biocompatibility risks associated with artificial prostheses . We previously demonstrated that autologous cartilaginous rods could be created in animal corpus cavernosum as penile prostheses in situ by transplanting autologous chondrocytes on biodegradable polymer scaffolds . In the present study we investigated the possibility of engineering human cartilage rods for potential use as penile prostheses . MATERIALS AND METHODS: Chondrocytes isolated from human ear were seeded on rod shaped biodegradable polymer scaffolds (1.2 cm . in diameter, 6.0 cm . long) . The seeded scaffolds were maintained in stirred bioreactors for 1 month . Subsequently, the seeded scaffolds were implanted subcutaneously into athymic rats . The specimens were retrieved 2 months after implantation, and histological, structural and mechanical properties were analyzed . The mechanical properties of the engineered prostheses were compared to those of silicone prostheses . RESULTS: Human chondrocytes seeded onto polymer scaffolds formed milk-white cartilaginous rods of the same size as the initial implants . Histological analyses using hematoxylin and eosin, toluidine blue and alcian blue showed mature and well-formed chrondrocytes in the retrieved implants . The engineered human cartilaginous rods were flexible, elastic and able to withstand high degrees of compressive forces . The mechanical properties were comparable to those of commercially available silicone prostheses . CONCLUSIONS: Transplantation of chrondrocytes isolated from human ear on polymer scaffolds resulted in the formation of human cartilage rods with the appropriate mechanical properties required for use as penile prostheses . This study demonstrates the feasibility of creating human cartilage rods with a large dimension . This technology may be useful for patients who need penile reconstruction.

Biotechnol Bioeng, 2002 Nov 20, 80(4), 465 - 76
RWPV bioreactor mass transport: earth-based and in microgravity; Begley CM et al.; Mass transport and mixing of perfused scalar quantities in the NASA Rotating Wall Perfused Vessel bioreactor are studied using numerical models of the flow field and scalar concentration field . Operating conditions typical of both microgravity and ground-based cell cultures are studied to determine the expected vessel performance for both flight and ground-based control experiments . Results are presented for the transport of oxygen with cell densities and consumption rates typical of colon cancer cells cultured in the RWPV . The transport and mixing characteristics are first investigated with a step change in the perfusion inlet concentration by computing the time histories of the time to exceed 10% inlet concentration . The effects of a uniform cell utilization rate are then investigated with time histories of the outlet concentration, volume average concentration, and volume fraction starved . It is found that the operating conditions used in microgravity produce results that are quite different then those for ground-based conditions . Mixing times for microgravity conditions are significantly shorter than those for ground-based operation . Increasing the differential rotation rates (microgravity) increases the mixing and transport, while increasing the mean rotation rate (ground-based) suppresses both . Increasing perfusion rates enhances mass transport for both microgravity and ground-based cases, however, for the present range of operating conditions, above 5-10 cc/min there are diminishing returns as much of the inlet fluid is transported directly to the perfusion exit . The results show that exit concentration is not a good indicator of the concentration distributions in the vessel . In microgravity conditions, the NASA RWPV bioreactor with the viscous pump has been shown to provide an environment that is well mixed . Even when operated near the theoretical minimum perfusion rates, only a small fraction of the volume provides less than the required oxygen levels . 2002 Wiley Periodicals, Inc.

Biotechnol Bioeng, 2002 Nov 20, 80(4), 438 - 44
Scale-up and optimization of an acoustic filter for 200 L/day perfusion of a CHO cell culture; Gorenflo VM et al.; Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited . A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein . The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics . Operational settings of the acoustic separator were optimized and the limits of robust operations explored . The performance was not influenced over wide ranges of duty cycle stop and run times . The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W . While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency . Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators . These results provide strong support for the feasibility of much greater scale-up of acoustic separations .

Biotechnol Bioeng, 2002 Nov 20, 80(4), 428 - 37
Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces; Ma N et al.; A microfluidic device was fabricated via photolithographic techniques which can create transient elongational and shear forces ranging over three orders of magnitude while still maintaining laminar flow conditions . The contractional fluid flow inside the microfluidic device was simulated with FLUENT (a computational fluid dynamics computer program) and the local deformation forces were characterized with the scalar quantity, local energy dissipation rate . The sensitivities of four cell lines (CHO, HB-24, Sf-9, and MCF7) were tested in the device . The results indicate that all four cell lines are able to withstand relatively intense energy dissipation rates (up to 10(4)-10(5) kW/m(3)), which is orders of magnitude higher than the maximum local energy dissipation rates generated by impellers in bioreactors, but comparable to that associated with small bursting bubbles . While the concept that suspended animal cells are relatively robust with respect to purely hydrodynamic forces in bioprocess equipment is well known, these results quantitatively demonstrate these observations .

Biotechnol Bioeng, 2002 Nov 20, 80(4), 405 - 13
Use of at-line and in-situ near-infrared spectroscopy to monitor biomass in an industrial fed-batch Escherichia coli process; Arnold SA et al.; One of the key goals in bioprocess monitoring is to achieve real-time knowledge of conditions within the bioreactor, i.e., in-situ . Near-infrared spectroscopy (NIRS), with its ability to carry out multi-analyte quantification rapidly with little sample presentation, is potentially applicable in this role . In the present study, the application of NIRS to a complex, fed-batch industrial E . coli (RV308/PHKY531) process was investigated . This process undergoes a series of temperature changes and is vigorously agitated and aerated . These conditions can pose added challenges to in-situ NIRS . Using the measurement of a key analyte (biomass) as an illustration, the details of the relationship between the at-line and in-situ use of NIRS are considered from the viewpoint of both theory and practical application . This study shows that NIRS can be used both at-line and in-situ in order to achieve good predictive models for biomass . There are particular challenges imposed by in-situ operation (loss of wavelength regions and noise) which meant the need for signal optimisation studies . This showed that whilst the at-line modelling process may provide some useful information for the in-situ process, there were distinct differences . This study shows that the in-situ use of NIRS in a highly challenging matrix (similar to those encountered in current industrial practice) is possible, and thus extends previous works in the area .

J Virol Methods, 2002 Sep, 105(2), 321 - 30
Quantitation of baculovirus particles by flow cytometry; Shen CF et al.; A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures . The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye . Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities . In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions . FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml . Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures . The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA) . Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA) . Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers . It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.

J Ind Microbiol Biotechnol, 2002 Sep, 29(3), 140 - 4
Continuous ethanol production in a nonconventional five-stage system operating with yeast cell recycling at elevated temperatures; Laluce C et al.; Three ranges of increasing temperatures (35-43, 37-45, 39-47 degrees C) were sequentially applied to a five-stage system continuously operated with cell recycling so that differences of 2 degrees C (between one reactor to the next) and 8 degrees C (between the first reactor at the highest temperature and the fifth at the lowest temperature) were kept among the reactors for each temperature range . The entire system was fed through the first reactor . The lowest values of biomass and viability were obtained for reactor R(3) located in the middle of the system . The highest yield of biomass was obtained in the effluent when the system was operated at 35-43 degrees C . This nonconventional system was set up to simulate the local fluctuations in temperature and nutrient concentrations that occur in different regions of the medium in an industrial bioreactor for fuel ethanol production mainly in tropical climates . Minimized cell death and continuous sugar utilization were observed at temperatures normally considered too high for Saccharomyces cerevisiae fermentations.

Biotechnol Appl Biochem, 2002 Oct, 36(Pt 2), 127 - 31
Application of statistically based experimental designs for the optimization of exo-polysaccharide production by Cordyceps militaris NG3; Xu CP et al.; Statistically based experimental designs were applied to the optimization of medium composition for exo-polysaccharide production by Cordyceps militaris NG3 in shake-flask cultures . First, the Plackett-Burman design was used to search for the main factors on mycelia and exo-polysaccharide production . Among these variables, sucrose and corn steep powder were found to be two significant factors and had positive effects on mycelial yield (with confidence level >80%) and exo-polysaccharide production (with confidence level >90%) . Subsequently, to study the mutual interactions between variables, the effects of the two main factors on exo-polysaccharide production were further investigated using a central composite design . The optimal composition was found to be 1.03 g/l corn steep powder, 2.95 g/l sucrose, 0.1 g/l K(2)HPO(4), 0.5 g/l MgSO(4) x 5H(2)O and 0.1 g/l KNO(3) for the enhanced production of the exo-polysaccharide, which was 2.604 g/l in shake-flask cultures . Under optimal culture conditions, the maximum exo-polysaccharide concentration in a 5 l stirred-tank bioreactor was 3.8 g/l.

Biotechnol Appl Biochem, 2002 Oct, 36(Pt 2), 95 - 100
Chlorpromazine oxidation by hydroperoxidase activity of covalent immobilized lipoxygenase; Santano E et al.; The application of the hydroperoxidase activity of immobilized lipoxygenase to xenobiotic metabolization is reported in this work . Soya bean lipoxygenase has been immobilized by covalent coupling to oxirane acrylic beads . This immobilized system has been applied to the oxidation of chlorpromazine (CPZ), a phenothiazine of wide pharmacological use which in some cases presents toxicity . Immobilized lipoxygenase produces the oxidation of CPZ in the presence of hydrogen peroxide at acidic pH, maintaining a high level of activity and stability . In comparison with free lipoxygenase, the immobilized enzyme system shows higher catalytic efficiency and protection against enzymic inactivation produced by the presence of H(2)O(2) . When the system of immobilized enzyme was loaded into a bioreactor operating in continuous mode, the level of CPZ oxidation was higher than obtained using a discontinuous procedure or the free enzyme . The results obtained in this work suggest that a system of covalent immobilized lipoxygenase operating in continuous mode may constitute a valuable tool for xenobiotic detoxification or metabolization studies.

J Chromatogr A, 2002 Aug 30, 968(1-2), 113 - 20
On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption; Noubhani AM et al.; In this report, we describe a new process for the on-line purification of His-tag EGFP (enhanced green fluorescent protein) taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption (IMAEBA) . The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms . This meets the needs of scientific researchers as well as industrialists interested in purifying recombinant proteins . After evaluating the different flow-rates and ultrasonic probe sizes, the on-line purification was tested . After ultrasonic treatment, 70% of the cells were broken and 90% of free EGFP was recovered after IMAEBA . In our conditions, more than 450 mg of EGFP were obtained in 15 h . On-line bioreactor-ultrasonic probe-immobilized metal affinity expanded bed adsorption is a rapid automated technique for obtaining large quantities of pure EGFP.

FASEB J, 2002 Oct, 16(12), 1691 - 4 Epub 2002 Aug 07.
Bioreactors mediate the effectiveness of tissue engineering scaffolds; Pei M et al.; We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three-dimensional (3D) scaffolds for cartilage tissue engineering . Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non-woven mesh, and composite woven/non-woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes) . Construct size, composition {cells, glycosaminoglycans (GAG), total collagen, and type-specific collagen mRNA expression and protein levels}, and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM*CS and SS*CS) were demonstrated . The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor-grown than dish-grown 3-day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor-grown than dish-grown 1-month constructs) . In bioreactors, mesh-based scaffolds yielded 1-month constructs with lower type I collagen levels and four-fold higher compressive moduli than corresponding sponge-based scaffolds . The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro-generated cartilage.

Hum Gene Ther, 2002 Sep 1, 13(13), 1621 - 30
Gene transfer of a human insulin-like growth factor I cDNA enhances tissue engineering of cartilage; Madry H et al.; The repair of articular cartilage lesions remains a clinical problem . Two novel approaches to cartilage formation, gene transfer and tissue engineering, have been limited by short-term transgene expression in transplanted chondrocytes and inability to deliver regulatory signals to engineered tissues according to specific temporal and spatial patterns . We tested the hypothesis that the transfer of a cDNA encoding the human insulin-like growth factor I (IGF-I) can provide sustained gene expression in cell-polymer constructs in vitro and in vivo and enhance the structural and functional properties of tissue-engineered cartilage . Bovine articular chondrocytes genetically modified to overexpress human IGF-I were seeded into polymer scaffolds, cultured in bioreactors in serum-free medium, and implanted subcutaneously in nude mice; constructs based on nontransfected or lacZ-transfected chondrocytes served as controls . Transgene expression was maintained throughout the duration of the study, more than 4 weeks in vitro followed by an additional 10 days either in vitro or in vivo . Chondrogenesis progressed toward the formation of cartilaginous tissue that was characterized by the presence of glycosaminoglycans, aggrecan, and type II collagen, and the absence of type I collagen . IGF-I constructs contained increased amounts of glycosaminoglycans and collagen and confined-compression equilibrium moduli as compared with controls; all groups had subnormal cellularity . The amounts of glycosaminoglycans and collagen per unit DNA in IGF-I constructs were markedly higher than in constructs cultured in serum-supplemented medium or native cartilage . This enhancement of chondrogenesis by spatially defined overexpression of human IGF-I suggests that cartilage tissue engineering based on genetically modified chondrocytes may be advantageous as compared with either gene transfer or tissue engineering alone.

Biotechnol Bioeng, 2002 Nov 5, 80(3), 349 - 57
Effect of particle-particle shearing on the bioleaching of sulfide minerals; Chong N et al.; The biological leaching of sulfide minerals, used for the production of gold, copper, zinc, cobalt, and other metals, is very often carried out in slurry bioreactors, where the shearing between sulfide particles is intensive . In order to be able to improve the efficiency of the bioleaching, it is of significant importance to know the effect of particle shearing on the rate of leaching . The recently proposed concept of ore immobilization allowed us to study the effect of particle shearing on the rate of sulfide (pyrite) leaching by Thiobacillus ferrooxidans . Using this concept, we designed two very similar bioreactors, the main difference between which was the presence and absence of particle-particle shearing . It was shown that when the oxygen mass transfer was not the rate-limiting step, the rate of bioleaching in the frictionless bioreactor was 2.5 times higher than that in a bioreactor with particle friction (shearing) . The concentration of free suspended cells in the frictionless bioreactor was by orders of magnitude lower than that in the frictional bioreactor, which showed that particle friction strongly reduces the microbial attachment to sulfide surface, which, in turn, reduces the rate of bioleaching . Surprisingly, it was found that formation of a layer of insoluble iron salts on the surface of sulfide particles is much slower under shearless conditions than in the presence of particle-particle shearing . This was explained by the effect of particle friction on liquid-solid mass transfer rate . The results of this study show that reduction of the particle friction during bioleaching of sulfide minerals can bring important advantages not only by increasing significantly the bioleaching rate, but also by increasing the rate of gas-liquid oxygen mass transfer, reducing the formation of iron precipitates and reducing the energy consumption . One of the efficient methods for reduction of particle friction is ore immobilization in a porous matrix .

Appl Microbiol Biotechnol, 2002 Sep, 59(6), 685 - 94 Epub 2002 Jul 17.
Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors; Miksch G et al.; The gene for phytase from Escherichia coli was sequenced and compared with the appA gene . It was found to be a mutant derivative of the appA gene . After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed . To develop a system for overexpression and extracellular production of phytase in E . coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed . Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level . An effective fermentation strategy based on fed-batch operation was developed.

Appl Microbiol Biotechnol, 2002 Sep, 59(6), 679 - 84 Epub 2002 Jul 13.
Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate; Bramucci M et al.; Sphingomonas strain ASU1 was isolated from an industrial wastewater bioreactor and grew on 2,6-dimethylnaphthalene (2,6-DMN) as the sole carbon/energy source . The genes for a xylene monooxygenase were cloned from strain ASU1 . Expression of the ASU1 xylene monooxygenase was compared to expression of the pWWO xylene monooxygenase in Escherichia coli . Both monooxygenases transformed p-xylene and 2,6-DMN by initially hydroxylating one methyl group . In addition, the ASU1 monooxygenase also hydroxylated the second methyl group on p-xylene and 2,6-DMN whereas the pWWO monooxygenase hydroxylated the second methyl group only on p-xylene . Endogenous E . coli enzymes contributed to further oxidation of the resulting aromatic alcohols to form aromatic carboxylates.

Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1473 - 8
Phenotype of hepatocyte spheroids in Arg-GLY-Asp (RGD) containing a thermo-reversible extracellular matrix; Park KH et al.; The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions . In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form) . A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells . In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc) . Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.

Planta Med, 2002 Aug, 68(8), 759 - 62
Features of development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides; Bondarev N et al.; Growth and development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides (SGs) were investigated . It was found that, owing to the highly favorable conditions of shoot cultivation created in such an apparatus, the intensity of shoot growth and SG production appeared to be 1.5 - 2.0 times higher than those of the shoots grown in tubes . These results indicate the existence of a positive correlation between these two processes . The data obtained suggest that the enhanced SG production is due to the differentiation of chlorenchyma cells and formation of specific subcellular structures for the glycoside to be accumulated.

Transpl Immunol, 2002 Aug, 10(2-3), 199 - 204
Monoclonal anti-D development programme; Kumpel BM; Administration of anti-D immunoglobulin to D- women after delivery of a D+ infant has dramatically reduced the number of immunised women and cases of haemolytic disease of the fetus and newborn . The use of monoclonal anti-D might alleviate some of the pressures on maintaining adequate supplies of plasma sourced anti-D . Two human monoclonal antibodies, BRAD-3 (IgG1) and BRAD-5 (IgG3), with proven activity in in vitro functional (immunological) assays with cells bearing IgG Fc receptors (Fc gammaR) were selected for clinical studies . They were prepared by purification of IgG secreted by culture of the Epstein-Barr virus-transformed B cell lines in hollow fibre bioreactors . The mean half-lives of BRAD-3 and BRAD-5 in D- subjects were 10.2 and 22.2 days, respectively . The clearance of D+ red cells injected into D- subjects was accelerated by prior injection of the monoclonal antibodies, both individually and blended (3:1, BRAD-5/BRAD-3) . The clearance rate was related to the amount of anti-D on the red cells . Clearance of the D+ red cells coated with BRAD-3 was more rapid in subjects homozygous for Fc gammaRIIIa-F/F158 than in those expressing the Fc gammaRIIIa-V158 allele . The subjects were protected from Rh D immunisation . A large multi-centre study evaluated the BRAD-3/5 blend for its ability to prevent Rh D immunisation in 95 D- subjects given 400 microg i.m . 24 h after injection of 5 ml D+ red cells . Challenge injections of D+ red cells alone were given 24 and 36 weeks later, and blood samples were taken every 4 weeks from the subjects throughout the study for detection of anti-D responses . There was one definite and one possible failure of protection; in one subject the plasma anti-D level rose from week 12 onwards, and in another individual rapid seroconversion was observed at week 28 . Considering the relatively large dose of red cells and the number of subjects studied, it was concluded that the failure rate was much lower than in routine Rh D prophylaxis . The responder rate was 13% by week 36 and 24% by week 48 . There was no relationship between HLA haplotype and Rh D immunisation . The low percentage of responders and the modest levels of endogenous anti-D produced suggested that administration of monoclonal anti-D had induced long-term specific suppression of anti-D responses in these subjects . The most likely mechanism of action was considered to be inhibition of B cells resulting from co-cross-linking antigen receptors with inhibitory Fc gammaR when the B cells contacted red cells that had bound passive anti-D.

Extremophiles, 2002 Aug, 6(4), 341 - 7 Epub 2002 Apr 18.
Trehalose production at high temperature exploiting an immobilized cell bioreactor; Di Lernia I et al.; The enzymatic production of trehalose from dextrins was studied as a series reaction in a packed bed reactor containing immobilized recombinant Escherichia coli cells, expressing either the Sulfolobus solfataricus (strain MT4) trehalosyl-dextrin forming enzyme (TDFE) or the trehalose-forming enzyme (TFE) . The cells, subjected to thermal treatments to increase cell permeability and to inactivate the unwanted host proteins, were entrapped separately or together in a calcium alginate polymeric matrix . The biocatalyst beads were used to pack a tubular glass reactor that was operated in a recycle mode . The performances of a bioreactor containing alternate layers of EcTFE and EcTDFE alginate beads were evaluated and compared with the performance of the co-immobilized biocatalysts . The latter showed a superior throughput, therefore the bioreactor packed with the co-entrapped biocatalysts was tested for the production of trehalose from concentrated dextrin solutions (10%-30% w/v) and a conversion up to 90% was obtained . This conversion corresponded to a production of 127 g trehalose h(-1) kg(-1) of biocatalyst . The results obtained suggest that the bioprocess described may be of interest in the development of a large-scale industrial process for trehalose production at high temperature.

J Environ Sci (China), 2002 Jul, 14(3), 406 - 12
Effect of leachate recycling and inoculation on the biochemical characteristics of municipal refuse in landfill bioreactors; Shen DS et al.; Activity development of key groups of enzymes involved in municipal refuse decomposition was measured in laboratory landfill bioreactors with and without leachate recycling and inoculation for about 210 days . The results showed that the enzymes (amylase, protease, cellulase, lipase and pectinase) were present in fresh refuse but at low values and positively affected by leachate recycling and refuse inoculation . The total average of cellulase activity in digesters D3 operated with leachate recycling but no inoculation, D4 and D5 operated with leachate recycling and inoculation was much higher than that in digesters D1 and D2 without leachate recycling and inoculation by 88%-127%, 117%-162% and 64%-98% . The total average of protease activity was higher in digester D4 than that in digesters D1, D2, D3 and D5 by 63%, 39%, 24% and 24%, respectively, and the positive effect of leachate recycling and inoculation on protease activity of landfilled refuse mainly was at the first two months . The total average of amylase activity was higher in digesters D3, D4 and D5 than that in digesters D1 and D2 by 83%-132%, 96%-148% and 81%-129% . During the early phase of incubation, the stimulatory effect of inoculation on lipase activity was measured, but refuse moisture was the main factor affecting lipase activity of landfilled refuse . The inoculation, initial and continuous inoculation of microorganisms existing in leachate, was the mainly stimulatory factor affecting pectinase activity of landfilled refuse.

Biotechnol Bioeng, 2002 Oct 5, 80(1), 114 - 22
The potential for establishment of axial temperature profiles during solid-state fermentation in rotating drum bioreactors; Mitchell DA et al.; The mixing and heat transfer phenomena within rotating drum bioreactors (RDBs) used for solid-state fermentation processes are poorly studied . The potential for the establishment of axial temperature gradients within the substrate bed was explored using a heat transfer model . For growth of Aspergillus oryzae on wheat bran within a 24 L RDB with air at a superficial velocity of 0.0023 m s(-1) and 15% relative humidity, the model predicts an axial gradient between the air inlet and outlet of 2 degrees C during rapid growth, compared to experimental axial temperature gradients of between 1 and 4 degrees C . Undesirably high temperatures occur throughout the bed under these operating conditions, but the model predicts that good temperature control can be achieved using humid air (90% relative humidity) at superficial velocities of 1 m s(-1) for a 204 L RDB . For a 2200 L RDB, good temperature control is predicted with superficial velocities as low as 0.4 m s(-1) with the airflow being switched from 90% to 15% relative humidity whenever the temperature at the outlet end of the drum exceeds the optimal temperature for growth . This work suggests that significant axial temperature gradients can arise in those RDBs that lack provision for axial mixing . It is therefore advisable to use angled lifters within RDBs to promote axial mixing .

Biotechnol Bioeng, 2002 Oct 5, 80(1), 1 - 12
Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor; Tay A et al.; A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae . Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability . The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions . A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation . When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)) . The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L . The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells . Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production . Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation . The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer .

Biotechnol Bioeng, 2002 Oct 20, 80(2), 156 - 68
Simulation of algae growth in a bench-scale bubble column reactor; Wu X et al.; The growth of the marine red microalga Porphyridium sp . in a bubble-column photobioreactor was simulated . The proposed model constitutes a dynamic integration of the kinetics of photosynthesis and photoinhibition with the fluid dynamics of the bubble column, including the effects of shear stress on the kinetics of growth . The kinetic data used in the model were obtained in independent experiments run in a thin-film photobioreactor with defined light/dark cycles . The maintenance term was modified to take into account the effects of liquid flow in the bioreactor on the growth rate . A hybrid method proposed for the approximate solution of the equations gave an appreciable reduction of the calculation time . Extrapolations of the model indicated the possibility of predicting the optimal diameter for an assembly of bubble column photobioreactors . Satisfactory fit was found with the experimental results of biomass growth in a 13-liter bubble column .

Biotechnol Bioeng, 2002 Oct 20, 80(2), 125 - 38
Development of a large-scale biocalorimeter to monitor and control bioprocesses; Voisard D et al.; Calorimetry has shown real potential at bench-scale for chemical and biochemical processes . The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor . To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation implemented to enable on-line monitoring and dynamic heat balance estimation . Providing that the signals are sufficiently precise, such a heat balance would enable calculation of the heat released or taken up during an operational (bio)process . Two electrical Wattmeters were developed, the first for determination of the power consumption by the stirrer motor and the second for determination of the power released by an internal calibration heater . Experiments were designed to optimize the temperature controller of the bioreactor such that it was sufficiently rapid so as to enable the heat accumulation terms to be neglected . Further calibration experiments were designed to correlate the measured stirring power to frictional heat losses of the stirrer into the reaction mass . This allows the quantitative measurement of all background heat flows and the on-line quantitative calculation of the (bio)process power . Three test fermentations were then performed with B . sphaericus 1593M, a spore-forming bacterium pathogenic to mosquitoes . A first batch culture was performed on a complex medium, to enable optimization of the calorimeter system . A second batch culture, on defined medium containing three carbon sources, was used to show the fast, accurate response of the heat signal and the ability to perfectly monitor the different growth phases associated with growth on mixed substrates, in particular when carbon sources became depleted . A maximum heat output of 1100 W was measured at the end of the log-phase . A fed-batch culture on the same defined medium was then carried out with the feed rate controlled as a function of the calorimeter signal . A maximum heat output of 2250 W was measured at the end of the first log-phase . This work demonstrates that real-time quantitative calorimetry is not only possible at pilot-scale, but could be readily applied at even larger scales . The technique requires simple, readily available devices for determination of the few necessary heat flows, making it a robust, cost-effective technique for process development and routine monitoring and control of production processes .

Water Sci Technol, 2002, 45(12), 159 - 65
A hybrid two-phase system for anaerobic digestion of food waste; Wang JY et al.; A hybrid two-phase system, consisting of a solid waste reactor as the acidification reactor and a wastewater reactor, i.e . an upflow anaerobic sludge blanket (UASB) reactor, as the methanogenic reactor, for anaerobic digestion of food waste was investigated . After the pre-acidification stage, COD and total VFA removals in the methanogenic phase were in the ranges of 74-93% and 77-100%, respectively, while leachate COD and total VFA concentrations in the acidification phase decreased by 95% and 97-99%, respectively . Some 99% of the total CH4 generated was from the methanogenic phase with the CH4 content of 68-70% . About 77-79% of TOC, 57-60% of volatile solids and 79-80% of total COD were removed . The results of this laboratory-scale study show that the hybrid two-phase anaerobic batch reactor system is suitable for effective conversion of food waste into CH4 and CO2 . The hybrid two-phase system can be further developed into an effective and efficient way to enhance waste stabilization in operating bioreactor landfills.

In Vitro Cell Dev Biol Anim, 2002 Apr, 38(4), 213 - 7
A countermeasure to ameliorate immune dysfunction in in vitro simulated microgravity environment: role of cellularnucleotide nutrition; Hales NW et al.; Considerable evidence suggests that space travelers are immunosuppressed, presumably by microgravity environmental stresses, putting them at risk for adverse effects, such as opportunistic infections, poor wound healing, and cancer . The purpose of this study was to examine the role and mechanisms of nucleotide (NT) supplementation as a countermeasure to obviate immunosuppression during space travel . The in vitro rotary cell culture system, a bioreactor (BIO), was used to simulate the effect of microgravity and to isolate the neuroendocrine effects inherent to in vitro models . The splenocytes from normal mice were cultured in BIO and control tissue culture (TC) flasks with and without phytohemagglutinin (PHA) for mitogen assays . The culture medium was then supplemented with various concentrations of a nucleosides-nucleotides mixture (NS + NT), inosine, and uridine . Cytokines interleukin (IL)-1beta, IL-2, IL-3, tumor necrosis factor-alpha, and interferon (IFN)-gamma were measured from the supernatant by enzyme-linked immunosorbent assay . In the PHA-stimulated cultures the cellular proliferation in the BIO was significantly decreased as compared with the TC flask cells . BIO-cultured cells in the presence of NS + NT maintained mitogen responses similar to the control TC flask cells . The maintenance of the mitogen response in BIO was observed by the supplementation of uridine and not of inosine . These results are in agreement with our earlier results from unit gravity experiments that showed that pyrimidines are more effective in pleiogenic immunoprotection to hosts . Cytokines IL-1beta, IL-2, and IFN-gamma in the BIO supernatants of cells cultured in the presence of NS + NT had a significantly higher response than the control vessel . Thus, supplemental NT, especially pyrimidines, can confer immune protection and enhance cytokine responses during space travel.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 360 - 4
{Continuous perfusion culture hybridoma cells for production of monoclonal antibody}; Mi L et al.; Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media . The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant . Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times . The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL . Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d . These results provided a primary mode of enlarge culture for monoclonal antibody industralization.

Water Sci Technol, 2002, 45(10), 219 - 24
Treatment of winery wastewater by an anaerobic sequencing batch reactor; Ruiz C et al.; Treatment of winery wastewater was investigated using an anaerobic sequencing batch reactor (ASBR) . Biogas production rate was monitored and permitted the automation of the bioreactor by a simple control system . The reactor was operated at an organic loading rate (ORL) around 8.6 gCOD/L.d with soluble chemical oxygen demand (COD) removal efficiency greater than 98%, hydraulic retention time (HRT) of 2.2 d and a specific organic loading rate (SOLR) of 0.96 gCOD/gVSS.d . The kinetics of COD and VFA removal were investigated for winery wastewater and for simple compounds such as ethanol, which is a major component of winery effluent, and acetate, which is the main volatile fatty acid (VFA) produced . The comparison of the profiles obtained with the 3 substrates shows that, overall, the acidification of the organic matter and the methanisation of the VFA follow zero order reactions, in the operating conditions of our study . The effect on the gas production rate resulted in two level periods separated by a sharp break when the acidification stage was finished and only the breaking down of the VFA continued.

Water Sci Technol, 2002, 45(10), 169 - 74
Retention of granular sludge at high hydraulic loading rates in an anaerobic membrane bioreactor with immersed filtration; Hernandez AE et al.; This study shows the results of an Anaerobic Membrane Bioreactor with immersed filtration cartridges for granular biomass retention working at high hydraulic loading rates . Biomass retention capabilities, granular biomass structure and activity, filtration cartridges flux and organic material degradation performance were evaluated . Four reactors with a volume of 0.005 m3 were tested . Two different filtration cartridges were used (10 and 100 microm) with effective surface area of 0.05 m2 . The filtration cartridges were submerged in the reactors for suction type filtration . Three organic loading rates were tested in the first two reactors and seven in the other two . Selective biomass washout and an increment in the biomass methanogenic activity was observed in the reactors at hydraulic loading rates between 1.2 and 4.5 m/h . Analyses of biomass granulometry showed an increase of larger size granular biomass . The immersed membrane anaerobic bioreactor maintain most of the advantages without biomass activity loss.

Aliment Pharmacol Ther, 2002 Aug, 16(8), 1547 - 54
Serum bile acids in patients with liver failure supported with a bioartificial liver; Pazzi P et al.; BACKGROUND: Serum bile acids are increased in liver failure, but the composition of the bile acid pool in this condition has not been studied in detail . This information is of interest because of dihydroxy bile acid toxicity . METHODS: We measured serum bile acids by gas chromatography-mass spectrometry in 13 patients with fulminant liver failure and five patients with acute-on-chronic liver failure . Furthermore, serum bile acids were analysed in the same patients after 6 h of treatment with a bioartificial liver, consisting of a hollow-fibre cartridge with microcarrier-attached porcine hepatocytes and a charcoal column . RESULTS: Pre-bioartificial liver serum bile acids demonstrated a high dihydroxy/trihydroxy ratio and were higher in patients with acute-on-chronic liver failure than in those with fulminant liver failure (452.8 +/- 98.6 vs . 182.1 +/- 39.7 micro mol/L; P < 0.05) . Bioartificial liver treatment decreased significantly serum bile acids in patients with fulminant liver failure (-38.8%) and acute-on-chronic liver failure (-35.8%), with a decreased dihydroxy/trihydroxy ratio . In vitro, porcine hepatocytes in the bioreactor cleared most conjugated bile acid species from pooled patient plasma . CONCLUSIONS: Acute liver failure is associated with very high serum levels of toxic bile acids that could contribute to the pathogenesis of the syndrome . Bioartificial liver treatment reduces both serum bile acid concentrations and the hydrophobicity of the bile acid pool.

Yi Chuan Xue Bao, 2002, 29(3), 206 - 11
{High expression of human FIX(hFIX) in transgenic mice directed by goat beta-casein gene promoter}; Huang Z et al.; To probe the feasibility of efficient production of human clotting factor IX(hFIX) with the approach of mammary gland bioreactor of transgenic animals, we constructed hFIX mammary gland expression vector containing promoter, exon 1, intron 1 and exon 2 of the goat beta-casein gene about 6.7 kb fragment as well as full-length of hFIX cDNA and its modified intron 1 sequence . By using transgenic products and 12 transgenic founders (9 female, 3 male) were produced, and the integration rate thus was 11.2% . ELISA assay and Western blot showed that the milk of 8 female transgenic mice had hFIX expression with high clotting activities . The highest hFIX expression in the milk of one transgenic mouse reached 52.9 mg/L, and the highest clotting activity of the transgene milk was 279.2% . FISH experiments indicated that hFIX DNA was integrated in different chromosomes in different mice . This result indicated that the hFIX mammary gland expression vector based on the goat beta-casein promoter can efficiently direct high expression of hFIX gene in the milk of transgenic mice, which maintained high clotting activity.

Curr Microbiol, 2002 Sep, 45(3), 203 - 8
Statistical media optimization and production of ITS alpha-amylase from Aspergillus oryzae in a bioreactor; Gigras P et al.; The production of an intermediate temperature-stable (ITS) alpha-amylase from Aspergillus oryzae was studied by using a central composite design with three independent variables, viz., starch, yeast extract, and K(2)HPO(4) . The model equation provided a suitable model for the response surface for alpha-amylase production, and, from the optimal concentrations of the medium components, a model was predicted, which was then used for enzyme production in a 150-L bioreactor . In the bioreactor studies, the enzyme yields (161 U/ml) were similar to that of the shake flask (133 U/ml); however, the time required for maximum alpha-amylase production in the bioreactor was reduced to 48 h compared with 120 h in shake flask cultures . An increased level of phosphate in the medium and low inoculum size were necessary to control the excessive foaming in the bioreactor; however, control of the pO(2) level and agitation was not mandatory for enzyme production . The peak enzyme production coincided with the increase in pH of the fermentation broth and was maximal when the pH of the system was above 7.5 . Thus, in the present study, pH acted as an indicator of the initiation or end of the enzyme synthesis or of the fermentation cycle.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 599 - 604 Epub 2002 Jun 22.
Adaptation of the white-rot basidiomycete Panus tigrinus for transformation of high concentrations of chlorophenols; Leontievsky AA et al.; During feed-batch cultivation of the white-rot fungus Panus tigrinus in a 5-l bioreactor on N-limited medium, 100, 200, 500, 1,000 and 2,000 mg 2,4,6-trichlorophenol (2,4,6-TCP) l(-1) were added sequentially after 90% removal of the previous portion of the toxicant . The addition of 500 mg 2,4,6-TCP l(-1) without preliminary adaptation killed the culture . The addition of 300 mg 2,4,6-TCP l(-1) without prior adaptation resulted in its slower removal than removal of 2,000 mg 2,4,6-TCP l(-1) by this adapted culture . After adaptation of P . tigrinus to 2,4,6-TCP in a 72-l bioreactor, the mixture of 2,4-dichlorophenol, 2,4,6-TCP, and pentachlorophenol, each at 500 mg x l(-1), was totally removed over 3 weeks . No lignin peroxidase activity was found in the course of cultivation of the fungus . Laccase activity was suppressed by addition of 2,4,6-TCP . Mn-peroxidase was found to be responsible for transformation of the chlorophenols . As final products of the process, several newly formed aromatic polymers, both chlorinated and non-chlorinated, were found in the culture liquid.

Mol Cell Proteomics, 2002 Jun, 1(6), 466 - 71
An improved protein bioreactor: efficient product isolation during in vitro protein biosynthesis via affinity tag; Lamla T et al.; In vitro protein biosynthesis became a powerful technology for biochemical research . Beside the determination of structure and function in vitro selection of proteins is also of great interest . In most cases the use of a synthesized protein for further applications depends on its purity . For this purpose the in vitro production and purification of proteins with short affinity tails was established . A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide . The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved . No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed . The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations . The results document that it should be possible to avoid these limitations by introducing an affinity system.

Tissue Eng, 2002 Jul, 8(3), 499 - 513
Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor; Powers MJ et al.; We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures . Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture . Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period . Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells . Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures . Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation . These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions . The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.

Tissue Eng, 2002 Jul, 8(3), 409 - 18
Optical measurement of three-dimensional collagen gel constructs by elastic scattering spectroscopy; Marenzana M et al.; Analysis of the formation and organization of new connective tissue formed in tissue-engineered constructs is a major requirement for tissue bioreactor technology . We have analyzed early-stage responses in collagen lattices, using elastic scattering spectroscopy to assess its potential to monitor tissue structural changes in structures up to 3 mm thick, under normal culture conditions . The method is based on an optical system in which an optical fiber delivers white light onto the tissue and the back-scattered light is collected for spectroscopy by another optical fiber . Results show correlation between changes in the spectral signatures with changes in the collagen gel contraction or internal organization in all three models of collagen construct analyzed . Therefore elastic scattering spectroscopy is a promising tool to monitor tissue-engineered constructs or early repair in collagenous tissues.

Cell Transplant, 2002, 11(4), 369 - 77
Hydrogel-coated textile scaffolds as three-dimensional growth support for human umbilical vein endothelial cells (HUVECs): possibilities as coculture system in liver tissue engineering; Risbud MV et al.; Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types . Here we report chitosan-collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs) . These scaffolds did not require precoating with fibronectin and they supported proper HUVEC attachment and growth . Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh . HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6) . MTT assay showed that cells had preserved mitochondrial functionality . It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds . Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds . Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix . Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor . Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells . We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

Ultrasonics, 2002 May, 40(1-8), 25 - 9
Ultrasound stimulation of micro-organisms for enhanced biodegradation; Schlafer O et al.; An ultrasound (US)-assisted bioprocess for wastewater treatment for the food industry was developed in a three years EC-funded project (FAIR CT-3259) . An improvement of biological activity by US was successfully demonstrated with lab scale reactors {Schlafer et al . Ultrasonics 38 (2000)} . Now a pilot scale optimisation with a 200 l bioreactor was carried out in co-operation with the Department of Industrial Acoustics, Technical University of Denmark and RESON A/S (Denmark) . Batch experiments have been performed by measuring the degradation rate of organic compounds of wastewater . The process parameters were kept constant while varying the US-intensity . A significant increase of biological activity could be obtained only in a very narrow range of US-intensity . Very low US-power leads to a decrease of degradation rate down to the standard value if there is no US-intake . Implementation of too high level of US lead to drastically decreasing of the biological activity to a level far below the Standard value if there is no US-intake . The optimisation of this narrow US-power window needs to be carried out carefully for every investigated bioprocess . The most important aspect of the experimental results was that a more than 100% increase of the maximum biological degradation rate has been achieved by US-transduction with an optimum US-intensity of 1.5 W/l wastewater at 25 kHz . The developed bioprocess reduces the overall energy by use of low-energy US-irradiation below the cavitation level . The US-assisted biological degradation process would be presented together with its optimisation and the economical calculation.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 898 - 903
Application of a variable structure model in observation and control of an anaerobic digestor; Tartakovsky B et al.; In this work, a variable structure model (VSM) of an anaerobic digestion process was developed . The anaerobic biodegradation process was described by four nonlinear submodels representing methanogenic, chemical oxygen demand overload, acidogenic, and hydrogen-inhibited states of the anaerobic process . At any instant, process dynamics was modeled only by one of the submodels, while the others were considered trailing . The choice of a leading submodel was handled by a knowledge-based system, which analyzed available process variables, such as off-gas composition and reactor pH . The feasibility of the proposed method was demonstrated both by using the VSM to predict the outputs of a comprehensive process model, and the experimental results obtained in a pilot scale anaerobic fixed-bed bioreactor.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 839 - 46
Cultivation of primary porcine hepatocytes in an OXY-HFB for use as a bioartificial liver device; Jasmund I et al.; Bioreactors being developed for bioartificial liver devices vary greatly in their construction . Until now, primary liver cells were cultivated either in sandwich configuration, as spheroids, or in special hollow fiber systems . Primary hepatocytes are demanding on their environment and have a high oxygen consumption . To get good results, optimal cultivation conditions are needed . The idea of the project was to investigate a new concept of an oxygenating hollow fiber bioreactor (OXY-HFB) . The OXY-HFB should consist exclusively of oxygenating and internal heat exchange fibers to yield a simple and effective design . Primary liver cells were seeded on the surface of the fibers in the extrafiber space . Oxygen requirements and temperature control were supplied through the fibers . The culture medium was perfused through the extrafiber space and therefore brought into direct hepatocellular contact . The OXY-HFB concept offers different advantages . A high cell density of 2.5 x 10(7) cells/mL can be obtained . This results in a cell number of 2.5 x 10(9) liver cells per bioreactor . Furthermore, the OXY-HFB is easily handled because no incubator is required . To study the efficiency of this bioreactor technique, various parameters were investigated over a cultivation period of three weeks . These included urea synthesis, lactate formation, glucose elimination, albumin synthesis, oxygen level, and pH . Furthermore, the metabolites of diazepam were measured . The biochemical performance of the bioreactor remained stable over the investigated time period . These results demonstrate that porcine liver cells preserve their viability and primary metabolism in the OXY-HFB over the complete period of study.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 776 - 81
An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter; Castilho LR et al.; In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process . A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments . In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations . The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced . In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands . An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system . The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles . After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out . No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 770 - 5
Biological conversion of anglesite (PbSO(4)) and lead waste from spent car batteries to galena (PbS); Weijma J et al.; Lead paste, a solid mixture containing PbSO(4), PbO(2), PbO/Pb(OH)(2) precipitate, and elemental Pb, is one of the main waste fractions from spent car batteries . Biological sulfidation represents a new process for recovery of lead from this waste . In this process the lead salts in lead paste are converted to galena (PbS) by sulfate-reducing bacteria . This paper investigates a continuous process for sulfidation of anglesite (PbSO(4)), the main constituent of lead paste, and lead paste, consisting of a laboratory-scale gas-lift bioreactor to which a slurry of anglesite or lead paste was supplied . Sulfate or elemental sulfur was added as an additional sulfur source . Hydrogen gas served as an electron donor for the biological reduction of sulfate and elemental sulfur to sulfide by sulfate- and sulfur-reducing bacteria . Anglesite was almost completely converted to galena at a loading rate of 19 kg of PbSO(4) m(-)(3) day(-)(1), producing a sludge of which the crystalline lead phases consisted of >98% PbS (galena) and 1-2% elemental Pb . With lead paste, stable sulfidation rates of up to 17 kg of lead paste m(-)(3) day(-)(1) were demonstrated, producing a sludge of which the crystalline lead phases consisted of an estimated >96% PbS, 1-2% elemental Pb, and 1-2% PbO(2).

Biotechnol Prog, 2002 Jul-Aug, 18(4), 752 - 9
Development of an optimal medium for continuous ferrous iron oxidation by immobilized Acidothiobacillus ferrooxidans cells; Kim TW et al.; This study was aimed at developing an immobilized bioreactor system in which long-term continuous ferrous iron oxidation can be realized with no formation of jarosite, which causes clogging of support pores and reactor lines . For this purpose, a medium with no jarosite formation was developed first by selecting optimal nitrogen and phosphate sources and their concentrations . Then with the developed medium containing ammonium phosphate instead of ammonium sulfate and potassium phosphate, repeated batch and continuous operations of ferrous iron oxidation by Acidothiobacillus ferrooxidans cells immobilized in a depth filter were successfully performed for an extended period of time . For about 510 h of operation including 450 h of continuous operation at dilution rates of 0.1, 0.2, and 0.3 h(-)(1), no formation of jarosite and thus no clogging of the reactor system were observed . The maximum ferrous iron oxidation rate was as high as 2.6 g/(L.h) at a dilution rate of 0.3 h(-)(1).

Water Environ Res, 2002 May-Jun, 74(3), 226 - 34
Entrapped mixed microbial cell process for combined secondary and tertiary wastewater treatment; Yang PY et al.; An entrapped mixed microbial cell (EMMC) process was investigated for simultaneous removal of carbon and nitrogen from a synthetic wastewater in a single bioreactor . The influent had a soluble chemical oxygen demand (SCOD)/nitrogen ratio varying from 4 to 15 and an alkalinity of 140 and 230 mg/L as calcium carbonate . An alternating schedule of intermittent aeration was used for two sizes of carriers: 10 x 10 x 10 mm3 and 20 x 20 x 20 mm3 . The medium carrier (10 x 10 x 10 mm3) was found to achieve higher removals of nitrogen, chemical oxygen demand, and 5-day biochemical oxygen demand of 92, 95, and 97%, respectively . These higher removal rates occurred at a 12-hour hydraulic retention time (HRT), an aeration/ nonaeration ratio of 0.5:2 hours, and an SCOD/nitrogen ratio of 15 in the influent . Influent alkalinity concentrations of 140 and 230 mg/L as calcium carbonate were found to have minimum effect on the removal of carbon and nitrogen . However, the oxidation-reduction potential, ranging from -100 to 400 mV (during air-off period), provides better nitrogen removal efficiency to maintain the total nitrogen (nitrate, nitrite, and ammonium) less than 10 mg/L when an SCOD/nitrogen ratio of 10 or 15 is maintained in the influent . Nitrogen removal efficiency increased with increasing ratios of SCOD/nitrogen in the influent (i.e., SCOD/ nitrogen ratio of 15 > 10 > 7 > 4.0) . The results of actual wastewater operation based on a study of synthetic wastewater show that organics removal efficiencies (94.6 and 94.2%) and nitrogen removal efficiencies (61.0 and 60.9%) are similar for reactors packed with medium and large carriers, respectively, when they are operated with a 9-hour HRT, an air on/air off ratio of 1 hour:2 hours and an SCOD/nitrogen ratio of 4.6.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 144 - 8
{Transgenic animals bioreactors}; Gou KM et al.; The production of human recombinant proteins in milk of transgenic farm animals offers a safe, very cost-effective source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods . This review has summarized the current status of gene selection, vector construct, transgenic methods, economics, and obvious potential in transgenic animals bioreactors . Recently, a more powerful approach was adopted in the transgenic animals founded on the application of nuclear transfer . As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic farm animals, product consistency, and time of product development . The successful adaptation of Cre-/lox P-mediated site-specific DNA recombination systems in farm animals will offer unprecedented possibilities for generating transgenic animals.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 136 - 9
{The progress of expressing vector for mammary gland bioreactor}; Chen HX et al.; The mammary gland bioreactor has great commercial value, but the examples for high level expression of foreign gene were so few that the most examples were not suitable for commercial program . The key resolution for improving the expression level of foreign gene is the construction of expression vector for mammary gland bioreactor . Nearly, many new ideas and new methods about the construction of expression vector were presented, the article summarized them.

Appl Environ Microbiol, 2002 Aug, 68(8), 3988 - 95
Life at the energetic edge: kinetics of circumneutral iron oxidation by lithotrophic iron-oxidizing bacteria isolated from the wetland-plant rhizosphere; Neubauer SC et al.; Batch cultures of a lithotrophic Fe(II)-oxidizing bacterium, strain BrT, isolated from the rhizosphere of a wetland plant, were grown in bioreactors and used to determine the significance of microbial Fe(II) oxidation at circumneutral pH and to identify abiotic variables that affect the partitioning between microbial oxidation and chemical oxidation . Strain BrT grew only in the presence of an Fe(II) source, with an average doubling time of 25 h . In one set of experiments, Fe(II) oxidation rates were measured before and after the cells were poisoned with sodium azide . These experiments indicated that strain BrT accounted for 18 to 53% of the total iron oxidation, and the average cellular growth yield was 0.70 g of CH2O per mol of Fe(II) oxidized . In a second set of experiments, Fe(II) was constantly added to bioreactors inoculated with live cells, killed cells, or no cells . A statistical model fitted to the experimental data demonstrated that metabolic Fe(II) oxidation accounted for up to 62% of the total oxidation . The total Fe(II) oxidation rates in these experiments were strongly limited by the rate of Fe(II) delivery to the system and were also influenced by O2 and total iron concentrations . Additionally, the model suggested that the microbes inhibited rates of abiotic Fe(II) oxidation, perhaps by binding Fe(II) to bacterial exopolymers . The net effect of strain BrT was to accelerate total oxidation rates by up to 18% compared to rates obtained with cell-free treatments . The results suggest that neutrophilic Fe(II)-oxidizing bacteria may compete for limited O2 in the rhizosphere and therefore influence other wetland biogeochemical cycles.

J Biotechnol, 2002 Sep 25, 98(2-3), 145 - 60
The methods to generate transgenic animals and to control transgene expression; Houdebine LM; Transgenic animals have been used for years to study gene function and to create models for the study of human diseases . This approach has become still more justified after the complete sequencing of several genomes . Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white . Improvement of animal production by transgenesis is still in infancy.Despite its intensive use, animal transgenesis is still suffering from technical limitations . The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated . The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA . The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes . Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals . Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

Artif Organs, 2002 Aug, 26(8), 710 - 4
Design of a new pulsatile bioreactor for tissue engineered aortic heart valve formation; Dumont K et al.; Evidence has been gathered that biomechanical factors have a significant impact on cell differentiation and behavior in in vitro cell cultures . The aim of this bioreactor is to create a physiological environment in which tissue engineered (TE) aortic valves seeded with human cells can be cultivated during a period of several days . The bioreactor consists of 2 major parts: the left ventricle (LV) and the afterload consisting of a compliance, representing the elastic function of the large arteries, and in series a resistance, mimicking the arterioles and capillaries . The TE aortic valve is placed between the LV and the compliance . With controllable resistance, compliance, stroke volume and frequency, and hydrodynamic conditions can be changed over a wide physiological range . This study resulted in a prototype of a compact pulsatile flow system for the creation of TE aortic valves . In addition a biocompatibility study of the used materials is performed.

Artif Organs, 2002 Aug, 26(8), 703 - 6
Modular extracorporeal liver support; Sauer IM et al.; Modular extracorporeal liver support (MELS) is an integrative concept for the treatment of hepatic failure with appropriate extracorporeal therapy units tailored to suit the actual clinical needs of each patient . The CellModule is a specific bioreactor charged with primary human liver cells harvested from human donor livers found to be unsuitable for transplantation due to steatosis, cirrhosis, or traumatic injury . The DetoxModule enables albumin dialysis for the removal of albumin-bound toxins, reducing the biochemical burden of the liver cells and replacing the bile excretion of hepatocytes in the bioreactor . A DialysisModule for continuous venovenous hemofiltration can be added to the system if required in hepatorenal syndrome.

Bioresour Technol, 2002 Sep, 84(2), 119 - 27
Recovery of resources for advanced life support space applications: effect of retention time on biodegradation of two crop residues in a fed-batch, continuous stirred tank reactor; Strayer RF et al.; Bioreactor retention time is a key process variable that will influence costs that are relevant to long distance space travel or long duration space habitation . However . little is known about the effects of this parameter on the microbiological treatment options that are being proposed for Advanced Life Support (ALS) systems . Two bioreactor studies were designed to examine this variable . In the first one, six retention times ranging from 1.3 to 21.3 days--were run in duplicate, 81 working-volume continuous stirred tank reactors (CSTR) that were fed ALS wheat residues . Ash-free dry weight loss, carbon mineralization, soluble TOC reduction, changes in fiber content (cellulose, hemicellulose, and lignin), bacterial numbers, and mineral recoveries were monitored . At short retention times--1.33 days--biodegradation was poor (total: 16-20%, cellulose - 12%, hemicellulose - 28%) but soluble TOC was decreased by 75-80% and recovery of major crop inorganic nutrients was adequate, except for phosphorus . A high proportion of the total bacteria (ca . 83%) was actively respiring . At the longest retention time tested, 21.3 days, biodegradation was good (total: 55-60%, cellulose ca . 70%, hemicellulose - ca . 55%) and soluble TOC was decreased by 80% . Recovery of major nutrients, except phosphorus, remained adequate . A very low proportion of total bacteria was actively respiring (ca . 16%) . The second bioreactor study used potato residue to determine if even shorter retention times could be used (range 0.25-2.0 days) . Although overall biodegradation deteriorated, the degradation of soluble TOC continued to be ca . 75% . We conclude that if the goal of ALS bioprocessing is maximal degradation of crop residues, including cellulose, then retention times of 10 days or longer will be needed . If the goal is to provide inorganic nutrients with the smallest volume/weight bioreactor possible, then a retention time of 1 day (or less) is sufficient.

Bioresour Technol, 2002 Aug, 84(1), 101 - 4
Long-term operation of slurry bioreactor for decomposition of food wastes; Park JI et al.; A pilot scale slurry bioreactor was used for the treatment of food wastes . Food wastes were continuously added (750 g wet weight per day) into the reactor and successfully decomposed to inorganic carbon without intermittent removal of suspended solids . During operation for 90 days, 91% reduction of food wastes was achieved . Microorganisms actively grew during the initial 20 days of operation, and reached a stationary phase with a cell concentration of around 5 x 10(10) cells ml(-1), which indicated that food waste was utilized as a respiratory substrate during this phase . Using data for time variation of dissolved oxygen, the oxygen requirement for decomposition of food wastes was estimated to be 5.0 g O2 g(-1) dry weight of food wastes.

Transplantation, 2002 Jul 15, 74(1), 13 - 21
Microgravity culture condition reduces immunogenicity and improves function of pancreatic islets1; Rutzky LP et al.; BACKGROUND: The failure of pancreatic islet allotransplants observed in almost all clinical attempts is related to poor initial islet function and allograft rejection . To remedy these problems we cultured islets in microgravity conditions to improve their function and to reduce their immunogenicity . METHODS: Fresh mouse islets or mouse islets cultured in stationary dishes or microgravity bioreactors were transplanted to streptozotocin-induced diabetic mouse recipients . RESULTS: Both allogeneic dish- or bioreactor-cultured islets survived more than 100 days compared with fresh allogeneic islets, which were rejected in less than 15 days . Islet titration studies revealed that 250 fresh or dish-cultured, but only 30 to 120 bioreactor-cultured, islets were necessary to produce euglycemia . Furthermore, glucose tolerance tests showed that bioreactor-cultured islets functioned better compared with fresh and dish-cultured islets on day 30 postgrafting . Immunostaining and transmission electron microscopy (TEM) analyses showed the gradual disappearance of dendritic cells in cultured islets compared with fresh islets . TEM revealed that the ultrastructure of islets from bioreactor, but not dish, appeared healthy and closely resembled fresh islets . Interestingly, TEM and scanning electron microscopy showed that only bioreactor-cultured islets developed unique and multiple nutritional channels between arrays of islet cells . TEM with colloidal lanthanum tracer revealed that only bioreactor islet cell cultures were devoid of tight junctional complexes, which may facilitate channel formation . CONCLUSION: Microgravity condition decreases immunogenicity and significantly improves the function of secretory cells.

Clin Nutr, 2002 Jun, 21(3), 231 - 8
Nutrition beyond nutrition: plausibility of immunotrophic nutrition for space travel; Kulkarni AD et al.; BACKGROUND AND AIMS: Microgravity has adverse effects on the immune system . We examined the effects of supplemental dietary nucleotides on immune function in ground-based in vivo anti-orthostatic tail-suspended (AOS) mice and in vitro (bioreactor-BIO) analogs of microgravity . METHODS: BALB/c mice were divided into the following three groups: group housed, single isolation, and AOS . Mice were fed either control chow or chow supplemented with RNA or uracil . Immune function was assessed by in vivo popliteal lymph node proliferation (PLN), in vitro PHA-stimulated proliferation of splenocytes and cytokine production . BIO splenocytes were cultured in vitro with/without PHA, a nucleoside-nucleotide mixture (NS/NT) or uridine . The cell proliferation and scanning electron microscopic examination for cells were carried out . RESULTS: PLN response was significantly suppressed in AOS mice (P<0.05) and was restored by RNA and uracil diets . Splenocytes from AOS mice had decreased phytohemagglutinin (PHA)-stimulated proliferation, decreased IL-2 and IFN-gamma cytokine levels (P<0.05) . These responses were restored by RNA and uracil diets . In BIO cultures, PHA response was suppressed significantly, and uridine and NS/NT restored the proliferative responses . Scanning electron microscopic analysis of cells cultured in BIO revealed cells with pinched, distorted and eroded membranes . Nucleotide supplementation especially uridine restored normal activated cell surface appearance and ruffling . CONCLUSION: In the microgravity analog environment of AOS and BIO, supplemental nucleotides and especially uracil/uridine have up-regulating and immunoprotective effects with potential as a countermeasure to the observed immune dysfunction in true microgravity.

Adv Biochem Eng Biotechnol, 2002, 76, 165 - 210
Chromatographic reactors based on biological activity; Podgornik A et al.; In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors . The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors . In this chapter, an overview of such chromatographic bioreactor systems is given . Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context.

Ann Bot (Lond), 2002 Jul, 90(1), 77 - 85
Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological, mineral and water characteristics; Barry-Etienne D et al.; Coffea arabica L . plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media . The intensity and quality of aerial and root system development were compared . Shoot emergence was more efficient in vitro but rooting frequencies were low . In contrast, all ex vitro-regenerated embryos rooted . The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro . Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets . Mortality was higher in nursery conditions, but better plant development was obtained . The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings . Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs . Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs . 233 mm-2) . Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s . 0.30 MPa) and relative water content values (97 vs . 93%) . Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised . Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.

Ann Bot (Lond), 2002 Jul, 90(1), 21 - 9
Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos; Afreen F et al.; Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF) . Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d . These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation . The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions . The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %) . Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied . Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel . The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor . Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor . Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel . In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.

J Biomed Mater Res, 2002 Oct, 62(1), 136 - 48
Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor; Sikavitsas VI et al.; The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix . MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells . The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture . On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP) . Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14 . The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18 . No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period . The spinner flask culture had the highest calcium content at day 14 . On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture . Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21 . This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds . The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds .

Ann Thorac Surg, 2002 Jul, 74(1), 46 - 52; discussion 52
Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells; Hoerstrup SP et al.; BACKGROUND: Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel . Particularly in a congenital operation, there is a substantial need for such implantation materials . We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells . Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated . METHODS: Human umbilical cord cells were harvested and expanded in culture . Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor . Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy . Characterization of extracellular matrix was comprised of immunohistochemistry . Extracellular matrix protein content and cell proliferation were quantified by biochemical assays . Biomechanical testing was performed using stress-strain and burst-stress tests . RESULTS: Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III . Cells stained positive for vimentin and alpha-smooth muscle actin . Scanning electron microscopy showed confluent, homogenous tissue surfaces . Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin . Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased . The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker . CONCLUSIONS: In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment . Morphologic and mechanical features approximated human pulmonary artery . Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.

Int J Artif Organs, 2002 Jun, 25(6), 542 - 8
A method to assess biochemical activity of liver cells during clinical application of extracorporeal hybrid liver support; Mundt A et al.; Biochemical activity of a hybrid liver support system based on porcine liver cells was investigated in patients suffering from acute liver failure, coma stage III-IV Patient plasma was drawn systemically and after circulation through the bioreactor at four hour intervals . A method is used that takes into account the rate of plasma flow and the differences in plasma concentration systemically and after circulation through the liver support system to determine the net release or uptake of metabolites such as ammonia, urea and glucose . Urea release (mean 2.28+/-0.37 micromol/h/g cells) and ammonia uptake (mean 0.17+/-0.11 micromol/h/g cells) was seen during treatment, an active role of the system in glucose metabolism was observed . All patients were bridged successfully to liver transplantation.

Biotechnol Bioeng, 2002 Aug 5, 79(3), 334 - 46
Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model; Travascio P et al.; A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E . coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA) . Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively . PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates . Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated . The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature . Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity . Temperature gradients were employed as a way to reduce the effects of diffusion limitation . Cephalexin was found to linearly increase with the applied temperature gradient . A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times . The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed .

Biotechnol Bioeng, 2002 Aug 5, 79(3), 277 - 83
Antibody production by a hybridoma cell line at high cell density is limited by two independent mechanisms; Gramer MJ et al.; Our previous attempt to model the stationary phase of production-scale hollow-fiber bioreactors using a scaled-down micro hollow-fiber bioreactor resulted in a predicted antibody production rate that was three- to fourfold lower than the actual value (Gramer and Poeschl, 2000) . Medium limitations were suspected as the reason for the discrepancy . In this study, various increases in medium feed rate were implemented in the micro bioreactor by increasing the diameter of the silicone tubing that houses the hollow fibers . Because larger diameter tubing may induce oxygen limitations, we also explored the effect of medium recirculation to enhance oxygenation . Antibody production in the micro bioreactor increased both as a result of increased medium supply and due to medium recirculation . However, these parameters increased antibody production through two independent mechanisms . The increased medium supply resulted in a higher cell-specific antibody production rate, but not a higher viable cell density . Medium circulation resulted in the support of a higher viable cell density, but had little effect on the cell-specific secretion rate . The two mechanisms of enhanced antibody production were additive, demonstrating that simultaneous parameters can limit antibody production by this cell line in a hollow-fiber system . When the medium feed and circulation rates were increased to a volumetrically proportional scale, scale-up predictions from the micro bioreactor matched the actual data from the production-scale system to within 15% . These data demonstrate the usefulness of the micro bioreactor for characterizing cell growth and limiting mechanisms at high cell densities .

Biotechnol Bioeng, 2002 Aug 20, 79(4), 416 - 28
A two-phase model for water and heat transfer within an intermittently-mixed solid-state fermentation bioreactor with forced aeration; Von Meien OF et al.; A two-phase dynamic model is developed that describes heat and mass transfer in intermittently-mixed solid-state fermentation bioreactors . The model predicts that in the regions of the bed near the air inlet there can be significant differences in the air and solid temperatures, while in the remainder of the bed the gas and solid phases are much closer to equilibrium, although there can be differences in water activity of around 0.05 . The increase in the temperature of the gas as it flows through the bed means that it is impossible to prevent the bed from drying out, even if saturated air is used at the air inlet . The substrate can dry to water activities that severely limit growth, unless the bed is intermittently mixed, with the addition of water to bring the water activity back to the desired value . Under the conditions assumed for the simulation, which was designed to mimic the growth of Aspergillus niger on corn, two mixing events were necessary, one at 17.4 and the other at 27.9 h . Even though such a strategy can minimize the restriction of growth by water-limitation, temperature-limitation remains a problem due to the rapid heating dynamics . The model is obviously a useful tool that can be used to guide scale-up and to test control strategies . Such a model, describing the non-equilibrium situation between the gas and solid phases, has not previously been proposed for solid-state fermentation bioreactors . Models in the literature that assume gas-solid temperature and moisture equilibrium cannot describe the large temperature differences between the gas and solid phase which occur within the bed near the air inlet .

Biotechnol Bioeng, 2002 Jun 5, 78(5), 545 - 55
Effect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I(2)B; Sahoo DK et al.; The influence of oxygen on glycerol production by an osmophilic yeast, Candida magnoliae I(2)B, was studied in a bioreactor . Oxygen transfer rates (OTRs) and volumetric oxygen transfer coefficients (k(L)a) were determined at different aeration and agitation rates . Cell growth as well as glycerol production was strongly affected by oxygen supply . Improvement in OTRs resulted in increased cell growth and glycerol yield . However, at high OTRs, there was a reduction in glucose uptake rate, indicating Pasteur Effect, and glycerol accumulation was also reduced at k(L)a of 253 h(-1) . The availability of oxygen per unit of cell mass was found to be the most important factor that controlled cell growth, glucose uptake, and glycerol yield . The overall productivity and yield of glycerol could be related with k(L)a . The biosynthesis of glycerol was found to both growth- and non-growth-associated, although glycerol was mainly produced in post-exponential phase .

Transgenic Res, 2002 Jun, 11(3), 257 - 68
Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by mammary-specific bovine alpha-lactalbumin regulation sequences; Chen CM et al.; Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII) . Hemophiliacs have been treated with whole plasma or purified FVIII concentrates . The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy . One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales . Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals . In the present study, a hybrid gene containing bovine alpha-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs . The alphaLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines . Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice . The concentrations of rFVIII ranged from 7.0 to 50.2 microg/ml, over 35-200-fold higher than that in normal human plasma . Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 368 - 76 Epub 2002 Apr 25.
Use of a two-phase partitioning bioreactor for degrading polycyclic aromatic hydrocarbons by a Sphingomonas sp; Janikowski TB et al.; A two-phase partitioning bioreactor (TPPB) utilizing the bacterium Sphingomonas aromaticivorans B0695 was used to degrade four low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAHs) . The TPPB concept is based on the use of a biocompatible, immiscible organic solvent in which high concentrations of recalcitrant substrates are dissolved . These substances partition into the cell-containing aqueous phase at rates determined by the metabolic activity of the cells . Experiments showed that the selected solvent, dodecane, could be successfully used in both solvent extraction experiments (to remove PAHs from soil) and in a TPPB application . Further testing demonstrated that solvent extraction from spiked soil was enhanced when a solvent combination (dodecane and ethanol) was used, and it was shown that the co-solvent did not significantly affect TPPB performance . The TPPB achieved complete biodegradation of naphthalene, phenanthrene, acenaphthene and anthracene at a volumetric consumption rate of 90 mg l(-1) h(-1) in approximately 30 h . Additionally, a total of 20.0 g of LMW PAHs (naphthalene and phenanthrene) were biodegraded at an overall volumetric rate of 98 mg l(-1) h(-1) in less than 75 h . Degradation rates achieved using the TPPB and S . aromaticivorans B0695 are much greater than any others previously reported for an ex situ PAH biodegradation system operating with a single species.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 239 - 45 Epub 2002 May 04.
High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter; Toksoy E et al.; Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia colicells . In system {R}, the TaqI endonuclease gene was cloned and expressed under the control of the strong T7 RNA polymerase promoter . To protect cellular DNA, methylase protection was provided by constitutive co-expression of TaqI methylase activity either by cloning the TaqI methylase gene on a second plasmid (system {R,M}) or by constructing a recombinant plasmid harboring both the endonuclease and methylase genes (system {R+M}) . In batch shake flasks containing complex media, co-expression of the methylase gene in systems {R,M} and {R+M} resulted in a 2- and 3-fold increase in volumetric productivity over system {R}, yielding activities of 250x10(6) U l(-1) and 350x10(6) U l(-1), which were 28 and 39 times higher than the data in the literature, respectively . Under controlled bioreactor conditions in chemically defined medium, co-expression of methylase activity greatly improved the yield and specific TaqI endonuclease productivity of the recombinant cells, and reduced acetic acid excretion levels . System {R,M} is preferable for high expression levels at longer operation periods, while system {R+M} is well-suited for high expression levels in short-term bioreactor operation.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 175 - 81 Epub 2002 Apr 20.
Inoculum production of the ectomycorrhizal fungus Pisolithus microcarpus in an airlift bioreactor; Rossi MJ et al.; Many important tree species in reforestation programs are dependent on ectomycorrhizal symbiosis in order to survive and grow, mainly in poor soils . The exploitation of this symbiosis to increase plant productivity demands the establishment of inoculum production methods . This study aims to propose an inoculum production method of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) using liquid fermentation in an airlift bioreactor with external circulation . The fungus grew as dark dense pellets during a batch fermentation at 25.5 degrees C and air inlet of 0.26-0.43 vvm . The maximum biomass (dry weight) achieved in the airlift bioreactor was approximately 5 g.l(-1) after 10-11 days . The specific growth rate (micro(x)) in the exponential phase was 0.576 day(-1), the yield factor (Y(X/S)) 0.418, and the productivity (P(X)) 0.480 g.l(-1).day(-1) . This specific growth rate was higher than that observed by other authors during fermentation processes with other Pisolithus isolates . The method seems to be very suitable for biomass production of this fungus . However, new studies on the fungus growth morphology in this system, as well as on the efficiency of the process for the cultivation of other ectomycorrhizal fungi, are necessary . It is also necessary to test the infectivity and efficiency of the inoculum towards the hosts.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 135 - 42 Epub 2002 Jun 11.
Industrial processes with animal cells; Kretzmer G; Industrial processes involving animal cells for the production of useful products still seem to be rather uncommon . Nevertheless, during the last four decades of the last century the number of relevant processes has increased from production of virus vaccines to monoclonal antibodies and finally complex structured glycoproteins . As soon as cell lines became permanent and culture medium changed from purely biological fluids to more or less defined chemical media, large-scale cultivation could begin . The developments of the 1970s - fusion of cells to form hybridomas, and genetic engineering - triggered a second wave of products . Monoclonal antibodies and recombinant proteins for diagnosis and therapy set new challenges for the inventors . Historically, there has been no straightforward process development since the product dictates the process operation . Therefore, the scale of production covers the whole range from small multiple-unit reactors (flasks or roller bottles) up to 10,000-l single-unit batch reactors . Products with high value and small demand can be produced in multiple-unit systems whereas "bulk" products for vaccination and therapy may need large-scale bioreactors to be cost effective . All the different systems have their advantages and disadvantages and significant challenges that curb the development of effective perfusion cultures still remain.

Water Res, 2002 May, 36(9), 2405 - 9
Effect of mass transfer on concentration wave propagation during anaerobic digestion of solid waste; Vavilin VA et al.; A distributed model of anaerobic digestion of solid waste was developed to study effects of mass transfer on the rate of propagation of initiation methanogenic area . The diffusion and advection of volatile fatty acids (VFA) and methanogenic biomass were taken into account in the model of a one-dimensional (ID) reactor . It was considered that VFA inhibits both polymer hydrolysis and acetoclastic methanogenesis . This approach allows to view the bioreactor as an active medium that provokes concentration waves from some area of methanogenic initiation (local VFA depression) to the total reactor volume . The model shows that mass transfer-based acceleration of methane production in the reactor is possible when the intensity of VFA utilization in the methanogenic area is sufficient for a complete digestion of the incoming VFA . Otherwise, initiation methanogenic area will be suppressed by increasing concentration of VFA . The obtained results emphasize the importance of considering spatial heterogeneity of the reaction for the analysis of solid anaerobic digestion in bioreactors and landfills . The digestion of solid waste can be optimized by setting the low rate of mass transfer (mixing or leachate recirculation) during lag-phase of the reaction with subsequent increase in the mass transfer rate in parallel with the propagation of methanogenic population . In this case, the rate of concentration waves substantially increases.

Am J Transplant, 2002 Mar, 2(3), 260 - 6
First clinical use of a novel bioartificial liver support system (BLSS); Mazariegos GV et al.; The first clinical use of the Excorp Medical Bioartificial Liver Support System (BLSS) in support of a 41-year-old African-American female with fulminant hepatic failure is described . The BLSS is currently in a Phase I/II safety evaluation at the University of Pittsburgh/UPMC System . Inclusion criteria for the study are patients with acute liver failure, any etiology, presenting with encephalopathy deteriorating beyond Parson's Grade 2 . The BLSS consists of a blood pump; a heat exchanger to control blood temperature; an oxygenator to control oxygenation and pH; a bioreactor; and associated pressure and flow alarm systems . Patient liver support is provided by 70-100 g of porcine liver cells housed in the hollow fiber bioreactor . The patient exhibited transient hypotension and thrombocytopenia at initiation of perfusion . The only unanticipated safety event was a lowering of patient glucose level at the onset of perfusion with the BLSS that was treatable with intravenous glucose administration . Moderate changes in blood biochemistries pre- and post perfusion are indicative of liver support being provided by the BLSS . While the initial experience with the BLSS is encouraging, completion of the Phase I/II study is required in order to more fully understand the safety aspects of the BLSS.

Water Res, 2002 Apr, 36(8), 2124 - 32
Kinetics of the aerobic biological degradation of shredded municipal solid waste in liquid phase; Liwarska-Bizukojc E et al.; The organic fraction of municipal solid waste (OFMSW) should be utilised by means of biological methods . The biodegradation of solid wastes can be intensified owing to application of the bioreactors . Estimation of the optimum values of the organic load is one of the most important tasks for the aerobic biodegradation processes . The kinetic model of biological oxidation of the organic wastes has been presented in this paper . The experiments were carried out in batch 6-l working volume stirred tank bioreactors at constant temperature of 25 degrees C . Initial total solids have been at the levels of 15, 19, 34, 55 and 66 g l(-1) . The kinetics of microbial decomposition of organic substances was described by means of an unstructured model . The satisfactory time courses for substrate chemical oxygen demand in the solid (CODs) and liquid phase (CODL) and biomass concentration (RNA) have been achieved . Also, the influence of the initial TS on the kinetics of the biodegradation process was investigated and the optimum value of initial TS for this type of processes was estimated at 34-55 g l(-1).

J Biotechnol, 2002 Aug 28, 97(3), 253 - 63
Optimization of feeding profile for a fed-batch bioreactor by an evolutionary algorithm; Ronen M et al.; The optimal feeding profile of a fed batch process was designed by means of an evolutionary algorithm . The algorithm chromosomes include the real-valued parameters of a profile function, defined by previous knowledge . Each chromosome is composed of the parameters that define the feeding profile: the feed rates, the singular arc parameters and the switching times between the profile states . The feed profile design was tested on a fed-batch process simulation . The accepted profiles were smooth and similar to those derived analytically in other studies . Two selection functions, roulette wheel and geometric ranking, were compared . In order to overcome the problem of model mismatches, a novel optimization scheme was carried out . During its operation the process was sampled, the model was updated and the optimization procedure was applied . The on-line optimization showed improvement in the objective function for relatively low sample times . Choosing the sampling frequencies depends on the process dynamics and the time required for the measurements and optimization . Further study on experiments of fed-batch process demonstrated the use of complex, non-differentiable model and produced improved process performances using the optimal feeding profile.

Biorheology, 2002, 39(1-2), 259 - 68
Bioreactor studies of native and tissue engineered cartilage; Vunjak-Novakovic G et al.; Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis . Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites . In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants . As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues . Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen . Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.

Ann N Y Acad Sci, 2002 Jun, 961, 210 - 5
Bioreactors and bioprocessing for tissue engineering; Ratcliffe A et al.; Bioreactor design in tissue engineering is complex, and at the early stages of its development . Design of biologically effective, yet scalable, devices requires intimate collaboration between engineers and biologists to ensure that all aspects are considered fully . Growth conditions, harvesting time, scale-up, storage, and sterility issues all need to be considered and incorporated into the design of bioreactors . Each tissue-engineered product will likely require individualized bioreactor design . However, without a comprehensive understanding of each of these components, bioreactor design and tissue growth to manufacture product will remain at a relatively rudimentary and limited level . Increased fundamental understanding of the issues can have a dramatic impact on the ability to generate tissue-engineered product safely, economically, and in the numbers that are required to fully address the patient populations in need.

Ann N Y Acad Sci, 2002 Jun, 961, 10 - 26
In vitro systems for tissue engineering; Godbey WT et al.; Tissue engineering, by necessity, encompasses a wide array of experimental directions and scientific disciplines . In vitro tissue engineering involves the manipulation of cells in vitro, prior to implantation into the in vivo environment . In contrast, in vivo tissue engineering relies on the body's natural ability to regenerate over non-cell-seeded biomaterials . Cells, biomaterials, and controlled incubation conditions all play important roles in the construction and use of modern in vitro systems for tissue engineering . Gene delivery is also an important factor for controlling or supporting the function of engineered cells both in vitro and post implantation, where appropriate . In this review, systems involved in the context of in vitro tissue engineering are addressed, including bioreactors, cell-seeded constructs, cell encapsulation, and gene delivery . Emphasis is placed upon investigations that are more directly linked to the treatment of clinical conditions.

Ann N Y Acad Sci, 2002 Jun, 961, 1 - 9
Tissue engineering and reparative medicine; Sipe JD; Reparative medicine is a critical frontier in biomedical and clinical research . The National Institutes of Health Bioengineering Consortium (BECON) convened a symposium titled "Reparative Medicine: Growing Tissues and Organs," which was held on June 25 and 26, 2001 in Bethesda, Maryland . The relevant realms of cells, molecular signaling, extracellular matrix, engineering design principles, vascular assembly, bioreactors, storage and translation, and host remodeling and the immune response that are essential to tissue engineering were discussed . This overview of the scientific program summarizes the plenary talks, extended poster presentations and breakout session reports with an emphasis on scientific and technical hurdles that must be overcome to achieve the promise of restoring, replacing, or enhancing tissue and organ function that tissue engineering offers.

J Pediatr Surg, 2002 Jul, 37(7), 1000 - 6; discussion 1000-6
Fetal tissue engineering: in utero tracheal augmentation in an ovine model; Fuchs JR et al.; BACKGROUND/PURPOSE: This study was aimed at comparing fetal tissue engineering with autologous free grafting in an ovine model of in utero tracheal repair . METHODS: Chondrocytes were isolated from both elastic and hyaline cartilage specimens harvested from fetal lambs and expanded in vitro . Cells were seeded dynamically onto biodegradable scaffolds, which then were maintained in a rotating bioreactor for 6 to 8 weeks . Constructs subsequently were implanted into fetal tracheas (n = 15), in a heterologous fashion (group I) . In group II, fetuses (n = 5) received autologous free grafts of elastic cartilage harvested from the ear as tracheal implants . In vivo specimens were harvested for histologic analysis at different time-points postimplantation . RESULTS: In the 12 of 15 surviving fetuses of group I, all constructs were found to resemble normal hyaline cartilage, engraft well despite their heterologous origin, and display time-dependent epithelialization derived from the native trachea . All autologous free grafts were engrafted and epithelialized at birth, retaining histologic characteristics of elastic cartilage, but were more deformed than engineered constructs . Of the lambs allowed to reach term, 5 of 5 in the engineered group and 4 of 5 in the free graft group could breathe spontaneously . CONCLUSIONS: (1) Tissue-engineered cartilage, as well as autologous free grafts, can be implanted successfully into the fetal trachea, resulting in engraftment and function . (2) Engineered cartilage provides enhanced structural support after implantation into the fetal trachea when compared with free grafts . Prenatal tracheoplasty may prove useful for the treatment of severe congenital tracheal malformations .

Biotechniques, 2002 Jun, 32(6), 1282 - 6, 1288
High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells; Howard MR et al.; Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning . Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells . The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium . Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium . Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated . Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible . The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

J Ind Microbiol Biotechnol, 2002 Mar, 28(3), 137 - 46
Production technology for entomopathogenic nematodes and their bacterial symbionts; Shapiro-Ilan DI et al.; Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria . The nematode-bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation . In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low . In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs . In vivo production and solid culture may be improved through innovations in mechanization and streamlining . In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced . Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design . A variety of formulations is available to facilitate nematode storage and application.

Artif Organs, 2002 Jun, 26(6), 497 - 505
A novel method for faster formation of rat liver cell spheroids; Okubo H et al.; Hepatocyte spheroids are expected to be the main component of the artificial liver bioreactor for their higher function . The preparation of hepatocyte spheroids, however, can require as many as 24 to 96 h . To reduce this time, we investigated a method employing a new technique of rat hepatocyte preparation and a dynamic culture . The modified Seglen's method for standard hepatocyte isolation was altered by elimination of ethyleneglycol bis(aminoethylether) tetraacetate from the first perfusate and calcium from the second perfusate . Isolated hepatocytes were cultured in a spinner flask by spinning at 120 rpm . The modified Seglen's method was used as a control . Cells obtained by the new method were more cohesive and formed a higher proportion of cell aggregates than control cells . In the spinning culture, hepatocytes had a tendency to aggregate and 80% of cells formed spheroids within 6 h of culturing . The mean size of spheroids was 68.5 +/- 18.5 microm . Confocal laser scanning microscopy revealed that individual spheroids contained approximately 30% of nonparenchymal cells over their surface . Using the new hepatocyte preparation method followed by a spinning culture, we were able to produce hepatocyte spheroids in as few as 6 h.

Hum Gene Ther, 2002 Jun 10, 13(9), 1075 - 80
The epidermis as a bioreactor: topically regulated cutaneous delivery into the circulation; Cao T et al.; Previous studies have documented that the skin can be used as a bioreactor to produce proteins for systemic release to treat diseases . A gene-switch system has been developed that allows regulated expression of therapeutic genes . To determine whether this system could be used in the skin, we developed a transgenic mouse model in which expression of a therapeutic gene could be topically induced in epidermal keratinocytes . After a single induction, high levels of the therapeutic protein, human growth hormone (hGH), were released from keratinocytes into the circulation . The serum levels of hGH were dependent on the amount of inducer applied, and repeated induction resulted in increased weight gain by transgenic versus control mice . Furthermore, physiological levels of hGH were detected in the serum of nude mice after topical induction of small transgenic skin grafts . These results clearly demonstrate the feasibility of using the gene-switch system to regulate the delivery of therapeutic proteins into the circulation via genetically modified keratinocytes.

J Biochem Biophys Methods, 2002 Apr 18, 51(2), 151 - 9
A cloth strip bioreactor with immobilized glucoamylase; D'Souza SF et al.; Glucoamylase was immobilized on polyethylenimine (PEI)-coated cotton cloth by adsorption followed by cross-linking with 0.2% glutaraldehyde in the presence of starch . Optimal adsorption of the enzyme was seen when cloth treated with 2% PEI was contacted with the enzyme for 50 min . pH and temperature optima profiles were not changed appreciably on immobilization . However, the bound enzyme exhibited a higher thermal stability . The enzyme-bound cloth strips were used in a specially designed bioreactor for the continuous hydrolysis of starch . The reactor could be operated for over 21 days retaining about 70% of the original activity . An operational temperature of 45 degrees C was found to be optimal.

ASAIO J, 2002 May-Jun, 48(3), 226 - 33
Plasma versus whole blood perfusion in a bioartificial liver assist device; Patzer JF 2nd et al.; The ramifications of using whole blood or plasma for perfusion off an hepatocyte containing bioartificial liver bioreactor in which the hepatocytes are separated by a membrane or other physical barrier from the perfusate stream on the rate of change of patient blood concentrations are explored through dynamic modeling of whole blood perfusion as a two compartment system (patient tissue and blood compartments), and plasma perfusion as a three compartment system (patient tissue and blood compartments, and a plasma reservoir) . The whole blood perfusion model is described by three dimensionless parameters: the Damkohler number, Da, which represents the ratio of the rate of conversion by the bioreactor to the rate of perfusion; kappa, which represents the ratio of the rate of internal reequilibration between the tissue and blood compartments and the rate of perfusion; and Vtb, the tissue/blood volume ratio . The plasma perfusion model has three additional dimensionless parameters: f, the fraction of plasma withdrawn from the blood in a plasma separator; alpha, the ratio of the plasma perfusion rate in the bioreactor to the blood draw rate; and Vbr, the blood/plasma reservoir volume ratio . Within the physiologic range of parameters, the rate of reduction in blood concentration in both the whole blood-perfused and plasma-perfused systems are sensitive to Damkohler number up to Da approximately 2 . Neither system is sensitive to variations in kappa, and the plasma perfusion system has little sensitivity to alpha . Given bioreactors of equivalent activity, a greater rate of blood concentration reduction and lower endpoint blood concentration at equivalent perfusion times will be achieved with whole blood perfusion . There are two physical reasons for this . The first is that the plasma perfused system is only processing a fraction, f, of the blood compared with the whole blood perfusion system . The second reason is that, although the blood-perfused system is limited by overall bioreactor performance, the plasma-perfused system is mass transfer limited to the rate of blood concentration dilution into the plasma reservoir rather than limited by the overall bioreactor performance.

Waste Manag Res, 2002 Apr, 20(2), 172 - 86
The bioreactor landfill: its status and future; Reinhart DR et al.; The bioreactor landfill provides control and process optimisation, primarily through the addition of leachate or other liquid amendments . Sufficient experience now exists to define recommended design and operating practices . However, technical challenges and research needs remain related to sustainability, liquid addition, leachate hydrodynamics, leachate quality, the addition of air, and cost analysis.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 572 - 7
Use of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase; Kundu B et al.; Understanding protein aggregation may hold important clues to understanding what goes wrong with protein folding in neurodegenerative disorders and in bioreactors in which proteins are overexpressed . Unfortunately, aggregates tend to be intractable to most standard methods of biochemical investigation . Thus, relatively little is even now known about the micro- and macro-structural features of aggregates . To gain insights into the thermal aggregation of a model globular protein {bovine carbonic anhydrase (BCA)}, we have used spectrofluorimetry to examine the binding of a hydrophobic dye, 8-anilinonaphthalene sulfonate (ANS), to hydrophobic clusters on the protein's surface both before and after heat-induced aggregation and upon cooling . Whereas native BCA shows no surface hydrophobicity, thermally aggregated BCA displays significant hydrophobicity both in the heated state and upon cooling . The timing of the addition of ANS in the course of aggregation makes no net difference to the ANS bound; we argue that this suggests that aggregates are essentially porous . Cooling of aggregates results in a dramatic, fully reversible increase in ANS binding that cannot be explained by the temperature dependence of fluorescence quantum yield alone; we argue that the enhancement of fluorescence upon cooling indicates possible structural consolidation of unfolded regions within aggregates (akin to refolding), with the required structural reorganization being facilitated by porosity . Finally, implications of porosity in aggregates are discussed, in particular, for the possible immobilization of enzymes through fusion with aggregation-prone protein domains.

Acta Astronaut, 2002 Jun, 50(12), 775 - 85
Novel aquatic modules for bioregenerative life-support systems based on the closed equilibrated biological aquatic system (C.E.B.A.S.); Bluem V et al.; The closed equilibrated biological aquatic system (C.E.B.A.S) is a man-made aquatic ecosystem which consists of four subcomponents: an aquatic animal habitat, an aquatic plant bioreactor, an ammonia oxidizing bacteria filter and a data acquisition/control unit . It is a precursor for different types of fish and aquatic plant production sites which are disposed for the integration into bioregenerative life-support systems . The results of two successful spaceflights of a miniaturized C.E.B.A.S version (the C.E.B.A.S . MINI MODULE) allow the optimization of aquatic food production systems which are already developed in the ground laboratory and open new aspects for their utilization as aquatic modules in space bioregenerative life support systems . The total disposition offers different stages of complexity of such aquatic modules starting with simple but efficient aquatic plant cultivators which can be implemented into water recycling systems and ending up in combined plant/fish aquaculture in connection with reproduction modules and hydroponics applications for higher land plants . In principle, aquaculture of fishes and/or other aquatic animals edible for humans offers optimal animal protein production under lowered gravity conditions without the tremendous waste management problems connected with tetrapod breeding and maintenance . The paper presents details of conducted experimental work and of future dispositions which demonstrate clearly that aquaculture is an additional possibility to combine efficient and simple food production in space with water recycling utilizing safe and performable biotechnologies . Moreover, it explains how these systems may contribute to more variable diets to fulfill the needs of multicultural crews . c2002 Elsevier Science Ltd . All rights reserved.

J Biotechnol, 2002 Jul 17, 97(1), 89 - 101
Mass transfer correlations for rotating drum bioreactors; Hardin MT et al.; Evaporative cooling is extremely important for large-scale operation of rotating drum bioreactors (RDBs) . Outlet water vapour concentrations were measured for a RDB containing wet wheat bran with the aim of determining the mass transfer coefficient for evaporation from the bran bed to the headspace . Mass transfer was expressed as the mass transfer coefficient times the area for transfer per unit volume of void space in the drum . Values of ka' were determined under combinations of aeration superficial velocities ranging from 0.006 to 0.017 ms(-1) and rotation rates ranging from 0 to 9 rpm . Mass transfer coefficients were evaluated using a variety of residence time distributions (RTDs) for flow in the gas phase including plug flow and well-mixed and a Central Jet RTD based on RTD studies . If plug flow is assumed, the degree of holdup at low effective Peclet (Pe(eff)) numbers gives an apparent under-estimate of ka' compared with empirical correlations . Values of ka' calculated using the Central Jet RTD agree well with values of ka' from literature correlations . There was a linear relationship between ka' and effective Peclet number: ka' = 2.32 x 10(-3)Pe(eff).

J Biotechnol, 2002 Jul 17, 97(1), 23 - 39
A fibrous-bed bioreactor for continuous production of developmental endothelial locus-1 by osteosarcoma cells; Chen C et al.; Genetically engineered human osteosarcoma cells containing developmental endothelial locus-1 (del-1) gene were studied for production of Del-1, a protein that has the properties of an extracellular matrix protein and can regulate vascular morphogenesis and remodeling . Del-1 has been studied as a potential anti-angiogenesis drug targeting solid tumors . In this study, osteosarcoma cells were cultured in a fibrous-bed bioreactor (FBB) to continuously produce Del-1 . The FBB was constructed by packing a polyester fibrous matrix into a 1.5-l spinner flask . The effects of media composition, including the serum content in the medium, and dilution rate on cell growth, metabolism, and Del-1 production were studied . A gradual reduction of serum content from 10% (v/v) to 0.5% (v/v) caused no loss in Del-1 production . However, the production of Del-1 decreased significantly in a serum-free medium, suggesting some nutrients present in the serum were important to culture viability and Del-1 production . The continuous FBB culture was stable for long-term production of Del-1, with a higher Del-1 titer than that normally obtained in T-flask cultures and overall productivity similar to the total production from 300 25-cm(2) T-flasks . Reducing geneticin in the medium from 250 microg ml(-1) to zero at later culturing stages had no significant effect on Del-1 production . The FBB was operated for a period of more than 4 months without any notable degeneration, and reached a final cell density of 3 x 10(8) cells ml(-1) of packing volume with >90% cell viability . The good reactor performance can be attributed to the three-dimensional environment provided by the fibrous matrix that allows for efficient mass transfer and cell immobilization and growth . Scanning electron microscopic and confocal scanning laser microscopic studies of the cell-matrix showed that cells formed large aggregates in the fibrous matrix and cell density was relatively uniform in the matrix.

J Biotechnol, 2002 Jul 17, 97(1), 13 - 22
Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding; Mendonca RZ et al.; The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes . In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers . Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture . Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities . Galactose was shown to be a more critical nutrient when cultures reached a high density . In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth . These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose . The inverse was not true and led, at high densities, to a decrease of cell viability . In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium . VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures . These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection . Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.

Biotechnol Prog, 2002 May-Jun, 18(3), 660 - 2
Olive oil mill waste waters decoloration and detoxification in a bioreactor by the white rot fungus Phanerochaete flavido-alba; Blanquez P et al.; Olive oil mill wastewater (OMW) is produced as waste in olive oil extraction . With the purpose of treating this highly polluting waste, a number of experiments were conducted in a laboratory-scale bioreactor with the white rot fungus Phanerochaete flavido-alba (P . flavido-alba) . It is known that this fungus is capable of decolorizing OMW in static or semistatic cultures at Erlenmeyer scale and at 30 degrees C . The objective of this work was to prove that P . flavido-alba could decolorize OMW in submerged cultures and that it is capable of reducing OMW toxicity at room temperature (25 degrees C) and in a laboratory-scale bioreactor . In the experiments conducted, manganese peroxidase (MnP) and laccase enzymes were detected; however, unlike other studies, lignin peroxidase was not found to be present . Decoloration obtained after treatment was 70% . The reduction of aromatic compounds obtained was 51%, and the toxicity of the culture medium was reduced by up to 70% . We can therefore state that P . flavido-alba is capable of reducing important environmental parameters of industrial effluents and that prospects are positive for the use of this process at a larger scale, even when working at room temperature.

Biotechnol Prog, 2002 May-Jun, 18(3), 501 - 8
Bioreactor production of human alpha(1)-antitrypsin using metabolically regulated plant cell cultures; Trexler MM et al.; Transgenic rice cell cultures, capable of producing recombinant human alpha(1)-antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor . The maximum specific growth rates (mu(max)) observed from two bioreactor runs were 0.40 day(-1) (doubling time of 1.7 days) and 0.47 day(-1) (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O(2)/(g dw h) . Using a metabolically regulated rice alpha-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium . After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability . Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium . Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day) . Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots.

Biotechnol Prog, 2002 May-Jun, 18(3), 445 - 50
Conversion of fumaric acid to L-malic by sol-gel immobilized Saccharomyces cerevisiae in a supported liquid membrane bioreactor; Bressler E et al.; Conversion of fumaric acid (FA) to L-malic acid (LMA) was carried out in a bioreactor divided by two supported liquid membranes (SLMs) into three compartments: Feed, Reaction, and Product . The Feed/Reaction SLM, made of tri-n-octylphosphine oxide (vol 10%) in ethyl acetate, was selective toward the substrate, fumaric acid (S(FA/LMA) = 10) . The Reaction/Product SLM, made of di(2-ethylhexyl) phosphate (vol 10%) in dichloromethane, was selective toward the product, L-malic acid (S(LMA/FA) = 680) . Immobilized yeast engineered to overproduce the enzyme fumarase {E.C . 4.2.1.2} was placed in the Reaction compartment and served as the catalyst . The yeast was immobilized in small glasslike beads of alginate-silicate sol-gel matrix . The construction of the bioreactor ensured unidirectional flow of the substrate from the Feed to the Reaction and of the product from the Reaction to the Product compartments, with the inorganic counterion traveling in the opposite direction . The conversion of almost 100%, above the equilibrium value of ca . 84% and higher than that for the industrial process, 70%, was achieved . In contrast to the existing industrial biocatalytic process resulting in L-malic acid salts, direct production of the free acid is described.

Appl Biochem Biotechnol, 2002 May, 101(2), 131 - 51
Optimization of glycerol fed-batch fermentation in different reactor states: a variable kinetic parameter approach; Xie D et al.; To optimize the fed-batch processes of glycerol fermentation in different reactor states, typical bioreactors including 500-mL shaking flask, 600-mL and 15-L airlift loop reactor, and 5-L stirred vessel were investigated . It was found that by reestimating the values of only two variable kinetic parameters associated with physical transport phenomena in a reactor, the macrokinetic model of glycerol fermentation proposed in previous work could describe well the batch processes in different reactor states . This variable kinetic parameter (VKP) approach was further applied to model-based optimization of discrete-pulse feed (DPF) strategies of both glucose and corn steep slurry for glycerol fed-batch fermentation . The experimental results showed that, compared with the feed strategies determined just by limited experimental optimization in previous work, the DPF strategies with VKPs adjusted could improve glycerol productivity at least by 27% in the scale-down and scale-up reactor states . The approach proposed appeared promising for further modeling and optimization of glycerol fermentation or the similar bioprocesses in larger scales.

Huan Jing Ke Xue, 2002 Mar, 23(2), 34 - 8
{The ecological succession of climax community restricted by COD/SO4(2-) ratio in acidogenic desulfate bioreactor}; Wang A et al.; The ecological succession of climax community restricted by COD/SO4(2-) ratio, the causing ecological factors, was investigated in acidogenic desulfate bioreactor with continuous-flow and batch-flow experiment . It was demonstrated that acetic acid type metabolization and acetic acid type climax community were the typical characteristic of acidogenic desulfate ecosystem, and the internal balance and feedback adjusting mechanism of the climax community was analyzed . In addition, three dimension realized ecological niche figure of preponderant population during the course of ecological succession was presented based on the following ecological factors of pH value, oxidation reduction potential and alkalinity.

Math Biosci, 2002 Jul-Aug, 179(1), 95 - 111
Optimal parametric sensitivity control for the estimation of kinetic parameters in bioreactors; Keesman KJ et al.; In this paper the well-known problem of optimal input design is considered . In particular, the focus is on input design for the estimation of kinetic parameters in bioreactors . The problem is formulated as follows: given the model structure (f,g), which is assumed to be affine in the input, and the specific parameter of interest theta;(k) find a feedback law that maximizes the sensitivity of the model output to the parameter under different flow conditions in the bioreactor and, possibly, minimize the input or state costs . Analytical solutions to these problems are presented . As an example a bioreactor with a biomass that grows according to the well-known Monod kinetics is considered.

J Environ Sci (China), 2002 Apr, 14(2), 181 - 7
Remove volatile organic compounds (VOCs) with membrane separation techniques; Zhang L et al.; Membrane separation, a new technology for removing VOCs including pervaporation, vapor permeation, membrane contactor, and membrane bioreactor was presented . Comparing with traditional techniques, these special techniques are an efficient and energy-saving technology . Vapor permeation can be applied to recovery of organic solvents from exhaust streams . Membrane contactor could be used for removing or recovering VOCs from air or wastewater . Pervaporation and vapor permeation are viable methods for removing VOCs from wastewater to yield a VOC concentrate which could either be destroyed by conventional means, or be recycled for reuse.

Water Res, 2002 Apr, 36(7), 1896 - 901
Biodegradation of a polymeric dye in a pulsed bed bioreactor by immobilised phanerochaete chrysosporium; Mielgo I et al.; An immobilised fungal bioreactor operated with pulsation of the gas-phase is proposed and operated at variable conditions for the continuous biological degradation of complex structures . In order to test the system, a hardly biodegradable dye (Poly R-478) was selected as a model compound and Phanerochaete chrysosporium as the ligninolytic fungus . High percentages of decolourisation--between 65% and 80%--under optimal conditions were achieved . Moreover, the system proved to have high stability with long operational periods (at least, 90 days) . During the operation Manganese Peroxidase was the sole ligninolytic enzyme detected which points out this enzyme as the main substance responsible for decolourisation . The optimal conditions established were the following: temperature of 37 degrees C, use of oxygen, Mn2+ concentrations between 33 and 330 microM and exogenous H2O2 added in periodical pulses (as chemical reagent, 1 microM or as H2O2 enzymatically generated by 0.6 U L(-1) glucose oxidase and residual glucose).

Water Res, 2002 Apr, 36(7), 1803 - 13
Comparison of the filtration characteristics of organic and inorganic membranes in a membrane-coupled anaerobic bioreactor; Kang IJ et al.; Comparison of filtration characteristics of organic and inorganic membranes was made in terms of physicochemical properties of the membrane materials, cake layer formation, backflushing and backfeeding effects in a membrane-coupled anaerobic bioreactor . For the inorganic membrane, struvite (MgNH4PO4 x 6H2O) was found to have accumulated inside the membrane pore and plays a key role in flux decline . For the organic, however, a thick cake layer composed of biomass and struvite formed on the membrane surface, thus causing a major hydraulic resistance . In order to mitigate flux decline for both membranes, backflushing and backfeeding modes were examined . With acidic (pH 2.0) backflushing, the flux was approximately doubled for the organic membrane . However, unexpectedly a negative effect was observed for the inorganic membrane . An alkaline backflushing instead of acidic backflushing gave rise to a flux improvement by a factor of two without any negative effect, even for the inorganic membrane . The backfeeding mode gave rise to a much higher flux compared with the normal mode in both types of membrane, although the flux returned to the same level as that with the normal mode after 6 days for the inorganic membrane . The differences between the two types of membranes were explained by membrane morphology, a ligand exchange reaction as well as a surface charge effect.

Int J Food Microbiol, 2002 May 25, 75(3), 231 - 9
An overview of small-scale food fermentation technologies in developing countries with special reference to Thailand: scope for their improvement; Valyasevi R et al.; Small-scale food fermentation technologies in developing countries have evolved through years of experience rather than through scientific breakthroughs . Many small-scale manufacturers are, therefore, reluctant to accept change and modify fermentation processes . Upgrading the quality and safety of fermented foods, while reducing their production cost and maintaining their authenticity and uniqueness, is of utmost importance . One strategic approach, which has been successfully implemented for the improvement of small-scale soy sauce fermentation in Thailand, is the consortium approach . This approach has allowed industry to work closely in sharing knowledge and common problems, which in turn has provided scientists with the research direction that would best benefit the industry . This consortium approach has brought about changes in the methodologies used in the production of soy sauce, by shortening processing times, introducing fiberglass tanks as bioreactors instead of the traditionally used small earthenware containers and introducing cost-effective waste management systems . One barrier to the application of starter cultures in the small-scale fermentation industry is the loss of uniqueness of fermented products . However, the advent of molecular biology techniques has allowed science to tailor starter cultures to the specific requirements of the manufacturer . Using the techniques of molecular biology, it has been shown that microflora of a specific product vary according to origin and sensory quality . A cell bank is being developed to serve as a resource base for Thai fermented pork sausage in order to facilitate the application of starter cultures in the manufacture of that product.

Can J Microbiol, 2002 Apr, 48(4), 333 - 41
Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products; Morgan CA et al.; The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle . Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively . Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients) . With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate . In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend . Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction . However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics . Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities . In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA . Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 627 - 40
Development of novel microscale system as immobilized enzyme bioreactor; Jones F et al.; This study involves a novel method for immobilized enzyme catalysis . The focus of the work was to design and construct a microscale bioreactor using microfabrication techniques traditionally employed within the semiconductor industry . Enzymes have been immobilized on the microreactor walls by incorporating them directly into the wall material . Fabricated microchannels have cross-sectional dimensions on the order of hundreds of micrometers, constructed using polydimethylsiloxane cast on silicon/SU-8 molds . The resulting ratio of high surface area to volume creates an efficient, continuous-flow reaction system . Transverse features also containing enzymes were molded directly into the channels.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 611 - 25
Bioreactor design studies for a hydrogen-producing bacterium; Wolfrum EJ et al.; Carbon monoxide (CO) can be metabolized by a number of microorganisms along with water to produce hydrogen (H2) and carbon dioxide . National Renewable Energy Laboratory researchers have isolated a number of bacteria that perform this so-called water-gas shift reaction at ambient temperatures . We performed experiments to measure the rate of CO conversion and H2 production in a trickle-bed reactor (TBR) . The liquid recirculation rate and the reactor support material both affected the mass transfer coefficient, which controls the overall performance of the reactor . A simple reactor model taken from the literature was used to quantitatively compare the performance of the TBR geometry at two different size scales . Good agreement between the two reactor scales was obtained.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 473 - 88
Hydrolysis of lactose by beta-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus: comparison of hollow-fiber membrane and packed-bed immobilized enzyme reactors for continuous processing of ultrahigh temperature-treated skim milk; Splechtna B et al.; Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli . Bioreactor cultivations of E . coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth . A technical-grade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80 degrees C . A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70 degrees C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof . CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70 degrees C . The packed-bed reactor was approximately 10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion . The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module . Under these bound conditions, CelB is essentially inactive . CelB is essentially inactive . Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 1037 - 48
Influence of a new axial impeller on K(L)a and xylanase production by Penicillium canescens 10-10c; Bakri Y et al.; The effects of a new axial impeller (HTPG4) on oxygen volumetric transfer coefficient, K(L)a, and xylanase production by Penicillium canescens 10-10c were studied and compared for dual-impeller systems, one with one DT4 impeller below and one HTPG4 above (DT4-HTPG4) and one with two DT4 (DT4-DT4) impellers, in a 5-L bioreactor . The volumetric coefficient of oxygen transfer was measured in culture medium using a gassing-out method at different gassing rates and agitation speeds . We observed that the DT4-HTPG4 combination provided better K(L)a performance than the DT4-DT4 combination . The two combinations were also tested for their influence on xylanase production by a filamentous microorganism; P . canescens 10-10c . These experiments demonstrated that the DT4-HTPG4 combination impeller enhanced enzyme production up to 23% compared with the DT4-DT4 combination at an aeration rate of 1 vvm and an agitation speed of 600 rpm . The main cause for this difference is thought to be a higher shear stress generated by the DT4-DT4 combination, which damages the mycelium of P . canescens and decreases xylanase production.

Appl Microbiol Biotechnol, 2002 May, 58(6), 756 - 60 Epub 2002 Mar 19.
Inclusion of solid particles in bacterial cellulose; Serafica G et al.; Depending upon the strain and the method of cultivation, bacterial cellulose can be reticulated filaments, pellets, or a dense, tough gel called a pellicle . The pellicular form is commonly made by surface culture, but a rotating disk bioreactor is more efficient and reduces the time of a run to about 3.5 days instead of the usual 12-20 days . Particles added to the medium as the gel is forming are trapped to form a new class of composite materials . Particles enter the films that are forming on the disks at rates depending on the size and geometry of the particle, as well as the rotational speed and concentration of the suspension.

Appl Microbiol Biotechnol, 2002 May, 58(6), 721 - 7 Epub 2002 Mar 07.
Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei; Bailey MJ et al.; Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques . Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media) . All the transformed strains exhibited vegetative growth properties similar to those of their parent . The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII . Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium . Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414 . Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30 . The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.

J Environ Sci Health B, 2002 May, 37(3), 265 - 75
Bacterial responses to temperature during aeration of pig slurry; Zhu J et al.; The temperature effect on total anaerobic and aerobic bacterial growth in pig slurry was studied using low level batch aeration treatments . Five bioreactors were built using Plexiglas tubes to perform five temperature treatments (5 degrees C, 10 degrees C, 15 degrees C, 20 degrees C, and 25 degrees C) . An airflow rate of 0.129 L/min/L manure was used to aerate manure contained in all reactors . Data showed that temperature had a profound impact on the aerobic counts in pig slurry during the aeration process . When the temperature increased from 15 degrees C to 25 degrees C, the average oxidation-reduction potential decreased from +40 mV to -60 mV, accompanied by a 75% reduction of aerobic bacteria in the manure . At 25 degrees C, the anaerobic counts were consistently higher than aerobic counts for most of days . A quadratic relationship was observed between the aerobic counts and the oxidation-reduction potential with a correlation coefficient of 0.8374 . To reduce odor generation potential, the oxidation-reduction potential in the manure should be maintained at +35 mV or higher.

J Biotechnol, 2002 May 23, 95(3), 195 - 204
Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells; Kallel H et al.; We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK . After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302 . The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS . Hence we have selected these media to study cell growth and the production of rabies virus . Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media . For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium . The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield . When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.

J Biomech Eng, 2002 Apr, 124(2), 258 - 61
A new flow chamber for the study of shear stress and transmural pressure upon cells adhering to a porous biomaterial; Chotard-Ghodsnia R et al.; Biomaterials used in some biomedical devices are porous and exposed to normal and tangential flow of biofluids . To examine the influence of flow induced forces on the morphology and the biochemical responses of cells adhering to such biomaterials, a Hele-Shaw cell with a porous bottom wall was designed and characterized experimentally . Theoretical predictions for the flow in the chamber are provided and allow to quantify the shear stress and/or transmural pressure exerted on cells . It is thus possible to follow up continuously the shape changes of cells that are adherent on a permeable membrane used in bioreactors.

J Chromatogr A, 2002 Mar 8, 949(1-2), 263 - 8
Monitoring of azo dye degradation processes in a bioreactor by on-line high-performance liquid chromatography; Rehorek A et al.; A technical solution and development of a method for on-line HPLC monitoring of bioreactor processes in a membrane reactor system are presented . Experiences in system design for the continuous coupling of a bioreactor system with capillary by-pass circuits using membrane flow cells and a dual HPLC system are reported . A continuously working integrated sample purification step by ultrafiltration with the membrane cell coupling is established . Using electrical switching valves and separated pumping and eluent systems, the dual HPLC system allows diode array detection as well as measurement of the refractive index . The application of the on-line HPLC monitoring system is demonstrated by measuring the anaerobic H-acid degradation kinetics . H-acid, 1-amino-8-hydroxynaphthalene-3,6-disulfonic acid, is one of the most important coupling components for a variety of direct, mordant, reactive dyes which remains in the process water and the textile dyeing effluents in high concentration.

Int J Artif Organs, 2002 Mar, 25(3), 192 - 202
Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients; Morsiani E et al.; Orthotopic liver transplantation (OLT) is the only effective therapeutic modality in severe acute hepatic failure (AHF) . The scarcity of organs for transplantation leads to an urgent necessity for temporary liver support treatments in AHF patients . A hepatocyte-based bioartificial liver (BAL) is under investigation with the main purpose to serve as bridging treatment until a liver becomes available for OLT, or to promote spontaneous liver regeneration . We developed a novel radial-flow bioreactor (RFB) for three-dimensional, high-density hepatocyte culture and an integrated pumping apparatus in which, after plasmapheresis, the patient's plasma is recirculated through the hepatocyte-filled RFB . Two hundred thirty grams of freshly isolated porcine hepatocytes were loaded into the RFB for clinical liver support treatment . The BAL system was used 8 times in supporting 7 AHF patients in grade III-IV coma, all waiting for an urgent OLT Three patients with no history of previous liver diseases were affected by fulminant hepatic failure (FHF) due to hepatitis B virus, 3 by primary non-function (PNF) of the transplanted liver, and one by AHF due to previous abdominal trauma and liver surgery . Six out of 7 patients underwent OLT following BAL treatment(s), which lasted 6-24 hours . All patients tolerated the procedures well, as shown by an improvement in the level of encephalopathy, a decrease in serum ammonia, transaminases and an amelioration of the prothrombin time, with full neurological recovery after OLT Our initial clinical experience confirms the safety of this BAL configuration and suggests its clinical efficacy as a temporary liver support system in AHF patients.

Chemosphere, 2002 Apr, 47(3), 325 - 32
As(III) removal from groundwaters using fixed-bed upflow bioreactors; Katsoyiannis I et al.; The application of biological oxidation of iron and manganese, as a potential treatment method for the removal of arsenic from contaminated groundwaters, was examined in this paper . This method was based on the growth of certain species of indigenous bacteria, which are capable of oxidizing the soluble iron and manganese ions; the oxidized forms can be subsequently removed from the aqueous stream by over 97%, through their transformation to insoluble oxides and separation by a suitable filter medium . Arsenic was removed by around 80%, under certain conditions, which were found to be sufficient for Fe(II) removal (dissolved oxygen 2.7 mg/l, redox 280-290 mV, pH 7.2, U 8.25 m/h) . The specific treatment technique presents several advantages towards conventional physicochemical treatment methods, such as enhanced coagulation or direct adsorption since: (a) it does not require the addition of other chemicals for oxidizing and removing As(III), (b) it does not require close monitoring of a breakthrough point, as in conventional column adsorption processes and (c) it could find application for the removal of, at least, three groundwater contaminants (Fe, Mn, As).

Biodegradation, 2001, 12(5), 291 - 301
Degradation of 2,3,4,6-tetrachlorophenol at low temperature and low dioxygen concentrations by phylogenetically different groundwater and bioreactor bacteria; Mannisto MK et al.; Effects of low temperature and low oxygen partial pressure on the occurrence and activity of 2,3,4,6-tetrachlorophenol degrading bacteria in a boreal chlorophenol contaminated groundwater and a full-scale fluidized-bed bioreactor were studied using four polychlorophenol degrading bacterial isolates of different phylogenetic backgrounds . These included an alpha-proteobacterial Sphingomonas sp . strain MT1 isolated from the full-scale bioreactor and three isolates from the contaminated groundwater which were identified as beta-proteobacterial Herbaspirillum sp . K1, a Gram-positive bacterium with high G + C content Nocardioides sp . K44 and an alpha-proteobacterial Sphingomonas sp . K74 . The Sphingomonas strains K74 and MT1 and Nocardioides sp . K44 degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol as the sole carbon and energy sources . Close to stoichiometric inorganic chloride release with the 2,3,4,6-tetrachlorophenol removal and the absence of methylation products indicated mineralization . Tetrachlorophenol degradation by the Herbaspirillum sp . K1 was enhanced by yeast extract, malate, glutamate, pyruvate, peptone and casitone . At 8 degrees C, Sphingomonas sp . K74 had the highest specific degradation rate (mu(max) = 4.9 x 10(-2) mg h(-1) cell(-1)) for 2,3,4,6-tetrachlorophenol . The Nocardioides strain K44 had the highest affinity (K(s) = 0.46 mg l(-1)) fortetrachlorophenol . K1 and MT1 grew microaerophilically in semisolid glucose medium . Furthermore, the growth of MT1 was inhibited in liquid glucose medium at high oxygen partial pressure indicating sensitivity to accumulating toxic oxygen species . On the other hand, trichlorophenol degradation was not affected by oxygen concentration (2-21%) . The isolates K44, K74 and MT1, with optimum growth temperatures between 23 and 25 degrees C, degraded tetrachlorophenol faster at 8 degrees C than at room temperature indicating distinctly different temperature optima for chlorophenol degradation and growth on complex media . These results show efficient polychlorophenol degradation by the isolates at the boreal groundwater conditions, i.e., at low temperature and low oxygen concentrations . Differences in chlorophenol degradation and sensitivities to chlorophenols and oxygen among the isolates indicate that the phylogenetically different chlorophenol degraders have found different niches in the contaminated groundwater and thus potential for contaminant degradation under a variety of saturated subsurface conditions.

Indian J Gastroenterol, 2002 Mar-Apr, 21(2), 55 - 8
In vitro studies on a bioreactor module containing encapsulated goat hepatocytes for the development of bioartificial liver; Khan AA et al.; BACKGROUND: A bioartificial liver may act as a temporary metabolic bridge in patients with acute liver failure . We devised a bioreactor module containing encapsulated goat hepatocytes and studied its efficiency in detoxifying ammonia . METHODS: A hollow-glass bioreactor module was designed and fabricated locally . The module was inoculated with alginate poly-L-lysine microcapsules with entrapped goat hepatocytes . Metabolism of ammonia and glucose was assessed . Antibody-mediated cell cytotoxicity was also assessed . RESULTS: The optimum encapsulated goat hepatocyte concentration was 12-18 billion at a perfusate flow rate of 30 mL/min under oxygenated condition . The optimum ammonium chloride concentration for detoxification was 2.5-5.0 mM . There was little or no cytolysis of encapsulated hepatocytes on exposure to complement-inactivated human AB serum . CONCLUSION: Encapsulated goat hepatocytes efficiently detoxified ammonia to urea . Cells were metabolically active up to 48 hours, indicating their feasibility for use in a bioreactor module . Encapsulation protected the hepatocytes from antibody-mediated cell lysis.

Biotechnol Bioeng, 2002 Jun 20, 78(6), 692 - 8
Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains; Calado CR et al.; This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)(2) and (WP)(4), respectively . Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains . The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg(-1) with activity per protein broth (specific activity) of 266 Umg(-1), whereas cutinase-(WP)(2) was secreted with a specific cell activity of 2.1 Umg(-1) with a specific activity of 200 Umg(-1), and cutinase-(WP)(4) with a specific cell activity of 0.7 Umg(-1) with a specific activity of 15 Umg(-1) . The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth . These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture) .

Biotechnol Bioeng, 2002 Jun 20, 78(6), 645 - 57
Control of starvation-induced apoptosis in Chinese hamster ovary cell cultures; Simon L et al.; The application of the unscented Kalman filter to control starvation-induced programmed cell death-apoptosis-in Chinese hamster ovary cells was investigated . Neural network-based sensitivity analysis identified glutamine and asparagine as two major amino acids that play a key role in the suppression of apoptosis . Dynamic equations that accounted for the dependence of apoptotic cells on the concentrations of viable cells, glutamine, and asparagine were derived . These state equations were highly nonlinear and included nine state variables . An oxygen mass balance was written in the liquid phase . It served as the output equation for the unscented Kalman filter . Using the oxygen uptake rate as the observer, it was possible to estimate the states . A model predictive controller was then implemented once the apoptotic cells in the bioreactor approached a concentration of 1.5 x 10(4) cells/mL, taking into account the operating range of the flow cytometer and measurement error . The manipulated variables were the flow rates of glucose, glutamine, and asparagine . Simulation results showed that the controller was able to keep the apoptotic cells at a concentration of 1.5 x 10(4) cells/mL .

Adv Biochem Eng Biotechnol, 2002, 74, 99 - 109
Bioreactor developments for tissue engineering applications by the example of the bioartificial liver; Jasmund I et al.; Tissue engineering is the application of the principles and methods of engineering and the life sciences towards the development of biological substitutes to restore, maintain or improve functions . It is an area which is emerging in importance worldwide . This article is to show the developments in tissue engineering research by the example of the bioartificial liver . As an alternative to liver transplantation, numerous researchers have been working towards the goal of development of a fully functional artificial liver . Liver support systems based on detoxification alone have proven ineffective because they cannot correct biochemical disorders . An effective artificial liver support system should be capable of carrying out the liver's essential processes, such as synthetic and metabolic functions, detoxification, and excretion . It should be capable of sustaining patients with fulminant hepatic failure and preparing patients for liver transplantation when a donor liver is not readily available . Although several hepatocyte-based liver support systems have been proposed, there is no current consensus on its eventual design configuration.

Adv Biochem Eng Biotechnol, 2002, 74, 183 - 206
Production of core and virus-like particles with baculovirus infected insect cells; Maranga L et al.; In this paper the fundamental aspects of process development for the production of core and virus-like particles with baculovirus infected insect cells are reviewed . The issues addressed include: particle formation and monomer composition, chemical and physical conditions for optimal cell growth, baculovirus replication and product expression, multiplicity of infection strategy, and scale-up of the process . Study of the differences in the metabolic requirements of infected and non-infected cells is necessary for high cell density processes . In the bioreactor, the specific oxygen uptake rate (OURsp) plays a central role in process scale-up, leading to the specification of the bioreactor operational parameters . Shear stress can also be an important variable for bioreactor operation due to its influence on cell growth and product expression . The determination of the critical variables in process development is discussed, showing the relevance of the mathematical models that have been developed for the insect cells/baculovirus system in process implementation and control.

Adv Biochem Eng Biotechnol, 2002, 74, 129 - 69
Cell retention devices for suspended-cell perfusion cultures; Castilho LR et al.; Perfusion cultures of animal cells have several advantages over batch or fed-batch cultures . They give, for instance, higher productivities and a consistent product quality, and allow steady state operation and better cell physiology control . However, one of the main aspects limiting performance and scale-up of perfusion processes is the need for an adequate cell retention device . The devices currently in use for stirred perfusion bioreactors are continuous centrifuges, tangential flow membrane filters, dynamic filters, spin-filters, ultrasonic and dielectrophoretic separators, gravity settlers and, more recently, hydrocyclones . The advantages and disadvantages of each of these methods will be discussed.

Adv Biochem Eng Biotechnol, 2002, 74, 111 - 28
Cultivation of hematopoietic stem and progenitor cells: biochemical engineering aspects; Noll T et al.; The ex vivo expansion of hematopoietic cells is one of the most challenging fields in cell culture . This is a rapidly growing area of tissue engineering with many potential applications in bone marrow transplantation, transfusion medicine or gene therapy . Over the last few years much progress has been made in understanding hematopoietic differentiation, discovery of cytokines, isolation and identification of cellular subtypes and in the development of a variety of bioreactor concepts . All this has led to a number of (preliminary) clinical trials that gave a hint of the benefits that can be obtained from the use of expanded hematopoietic cells in therapy . Moreover, as we understand the complexity and the regulation of hematopoiesis, it becomes obvious that highly sophisticated cultivation techniques and bioreactor concepts are needed: a new challenge for bioprocess engineering in cell culture.

Bioresour Technol, 2002 May, 82(3), 225 - 31
Anaerobic decolorisation of simulated textile wastewater containing azo dyes; Manu B et al.; This study deals with the decolorization of the commercially important azo dyes, Orange II (C.I . Acid Orange 7) and Reactive Black 3HN (C.I . Reactive Black 8) under anaerobic conditions in wastewater . Laboratory scale semicontinuous studies were conducted using simulated cotton dyeing wastewater at ambient temperatures (24-28 degrees C) by maintaining a HRT of 10 days . The dye concentration in wastewater was maintained at 100 mg/l . The reactors were operated for 58 days and Orange II and Black 3HN were easily decolorized under the experimental conditions employed . The performance of the bioreactors was evaluated by monitoring oxidation-reduction potential (ORP) in the reactor, color and Chemical oxygen demand (COD) removal . Color removal of >99% was achieved in both the dye-containing reactors . COD removals of up to 95%, 92% and 94% were achieved in control, orange- and black dye-containing reactors, respectively . Effect of dyes and salts present in textile wastewater on methanogenesis was evaluated based on maximum methane production and methanogenic activity . Based on the maximum methane production data, no inhibition of methanogenesis was observed for dye concentrations of up to 400 mg/l for both the dyes . However from the methanogenic activity data, it was observed that the black dye concentration of 400 mg/l seemed to cause inhibition of methanogenesis.

Vestn Ross Akad Med Nauk, 2002, (3), 21 - 3
{Examining the properties of hepatic cytosol and the efficiency of its use in the Biological Support Liver apparatus}; Riabinin VE et al.; Clinical trials showed the minimum efficiency of various detoxifying methods (dialysis, charcoal adsorption, plasma exchange) in terms survival in acute hepatic insufficiency . Attempts were made to provide hepatic functions through ex vivo perfusion of the whole liver and tissue sections, but these methods remain a challenge to immune responses and blood clotting . This led to the development of the extracorporeal liver support systems and early clinical results encouraged further work in this area . The liver support system consists of a bioreactor containing a liver homogenate, a transfer mechanism, and a dialyzer that connects 2 closed contours . The apparatus is connected to the patient's blood system by means of an arteriovenous or venovenous shunt . The contact time of a liver homogenate and a patient's blood in the dialyzer and the diffusion rate of molecules of chemical substances determine the detoxifying and metabolic effects . Laboratory studies have demonstrated that the detoxifying activation method has high effects (increased biotransformation, decreased blood ammonia levels), decreases the intensity of pathological processes, normalizes blood components (higher levels of ATP, glucose, amino acids, vitamin C) . Model and experimental studies of the efficiency of the method lead to the conclusion that it is expedient to apply it in medical practice.

Planta Med, 2002 Apr, 68(4), 336 - 40
Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana; Navia-Osorio A et al.; A cell suspension culture of Taxus wallichiana (Himalayan Yew) was grown in shake flasks and a 20-L airlift bioreactor running for 28 days in a batch mode, and its capacity to accumulate paclitaxel and baccatin III was measured . When both culture types were in the highest productive state (from day 24 to day 28), there was a greater accumulation of paclitaxel and baccatin III in the bioreactor culture than in the shake flask culture (factor of 2.0 and 1.2, respectively) . These increases in paclitaxel and baccatin III production cannot be related to the difference observed between the growth rates of both cultures, because when the bioreactor culture was at maximum productivity, its cell biomass, expressed in g L(-1) of dry weight, was similar to that obtained in the shake flask culture . It seems that these improvements were mainly due to adequate aeration and mixing of the culture in the bioreactor . The maximum yield observed for paclitaxel (20.84 mg x L(-1) day 24) and baccatin III (25.67 mg x L(-1) day 28) represents a productivity of 0.90 mg x L(-1) d(-1) and 0.93 mg x L(-1) x d(-1) respectively.

J Immunol Methods, 2002 Apr 1, 262(1-2), 71 - 83
Ex vivo expansion of functional T lymphocytes from HIV-infected individuals; Trickett AE et al.; This study was designed to define the conditions for expansion of functional T lymphocytes from human immunodeficiency virus (HIV)-infected subjects, with the ultimate goal of using these cells for immunotherapy . The most appropriate culture conditions for good T cell proliferation included stimulation with anti-CD3 and anti-CD28 coated microspheres, and propagation in Aim V serum-free media with 20 U/ml interleukin-2 (IL-2), supplemented with decreasing concentrations of serum for the initial 8 days . Under these conditions, a 14-day culture period yielded approximately a 10,000-fold expansion of T lymphocytes from HIV-infected donors . The cultured cells comprised approximately 15% CD4+ cells and 70% CD8+ cells . These cells retained functional capacity as assessed by cytotoxicity towards HIV proteins, and production of IL-2 and interferon-gamma (IFN-gamma) . Viral replication within the culture system was controlled, but not eliminated, without the requirement for antiviral agents . These culture conditions were demonstrated to be suitable for larger scale expansion of cells in hollow fibre bioreactors . This methodology provides a suitable means of producing large quantities of functional T cells for use in autologous immunotherapy protocols.

Nat Biotechnol, 2002 May, 20(5), 484 - 7
Large scale production of recombinant human lactoferrin in the milk of transgenic cows; van Berkel PH et al.; The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems . The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins . Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk . Natural hLF from human milk and rhLF had identical iron-binding and -release properties . Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection . Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.

Ann Pharm Fr, 2002 Jan, 60(1), 28 - 37
{Biosensors in the pharmaceutical domain}; Kauffmann JM; Biosensors are analytical devices which incorporate a biological component (enzyme, antibody, animal or plant cell, DNA fragments, lipids.) intimately connected to a physical transducer (electrode, optical fibre, vibrating quartz.) . This dual configuration allows the study of a great variety of compounds of pharmaceutical interest which react with the biocomponent . The latter is selected depending on the application and the performance criteria requested . Biosensors are suitable for real time monitoring such as in bioreactors, and for the determination of various physiological and pharmacological parameters . Biosensors may be employed in home testing (glucose, lactate.), in hospitals (bedside testing, emergency, surgery, dialysis monitoring, etc.) in clinical laboratory analyses (immunoassays, DNA analysis.) and at research centres . Ideally, a biosensor should be easy to use, allowing direct analysis without sample pre-treatment . Measurements should be automatized and remote controlled . The biosensor may be miniaturized for single use or for implementation in sensor arrays . Applications to microenvironments (in vivo, single cell.) or discrete one shot decentralized tests may also considered.

Lett Appl Microbiol, 2002, 34(5), 370 - 5
Improvement for the production of clavulanic acid by mutant Streptomyces clavuligerus; Lee SD et al.; AIMS: To improve the production of clavulanic acid through the development of strains, the selection of a production medium and a pH shift strategy in a bioreactor . METHODS AND RESULTS: Streptomyces clavuligerus mutant 15 was selected by antibacterial activities . As a result of pH control in a 2.5 l bioreactor, the highest productivity (3.37 microg x ml(-1) x h(-1)) was obtained at a controlled pH of 7.0 . CONCLUSIONS: The highest level of production obtained was an increase of about 36% compared with a non-controlled pH . When the production of clavulanic acid reached the maximum level, the pH was shifted from 7.0 to 6.0 for reduction in decomposition rate . The maximum concentration of clavulanic acid was maintained for 24 h as a result of the pH shift control, and a significant reduction in the decomposition of clavulanic acid was obtained . SIGNIFICANCE AND IMPACT OF THE STUDY: Clavulanic acid decomposition was considerably reduced as a result of the pH shift control . The results of this study can be applied for the efficient production of beta-lactamase inhibitory antibiotics.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 537 - 49
Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor; Almeida RM et al.; Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess . Other alternatives for the production of beta-lactam antibiotics, such as a cephalosporin C fed-batch process in an aerated stirred-tankbioreactor with free cells of Cephalosporium acremonium, or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch process . In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium . In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations . For this study's model, modifications were introduced to take into account the influence of supplementary medium flow rate . The balance equations considered the effect of oxygen limitation inside the bioparticles . In the Monod-type rate equations, cell concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate . The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval . The simulation results showed that the proposed model fit well with the experimental data, and based on the

Appl Biochem Biotechnol, 2001 Spring, 91-93, 341 - 52
A hybrid neural network algorithm for on-line state inference that accounts for differences in inoculum of Cephalosporium acremonium in fed-batch fermentors; Silva RG et al.; One serious difficulty in modeling a fermentative process is the forecasting of the duration of the lag phase . The usual approach to model biochemical reactors relies on first-principles, unstructured mathematical models . These models are not able to take into account changes in the process response caused by different incubation times or by repeated fedbatches . To overcome this problem, we have proposed a hybrid neural network algorithm . Feedforward neural networks were used to estimate rates of cell growth, substrate consumption, and product formation from on-line measurements during cephalosporin C production . These rates were included in the mass balance equations to estimate key process variables: concentrations of cells, substrate, and product . Data from fed-batch fermentation runs in a stirred aerated bioreactor employing the microorganism Cephalosporium acremonium ATCC 48272 were used . On-line measurements strongly related to the mass and activity of the cells used . They include carbon dioxide and oxygen concentrations in the exhausted gas . Good results were obtained using this approach.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 311 - 6
Effect of yeast extract on growth kinetics of Monascus purpureus; Pereira DG et al.; Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied . A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a two-step cultivation in a shaker . First, the spores were cultivated in a complex medium rendering a suspension of vegetative cells . In the second step these cells were grown in a semisynthetic medium . Two types of media were employed in the bioreactor runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract . The inclusion of yeast extract, caused an increase in cell yield on glucose (Yx/s) as high as 40% . Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative to the synthetic medium . On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble pigments.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 283 - 96
Ethanol production in a membrane bioreactor: pilot-scale trials in a corn wet mill; Escobar JM et al.; Pilot plant trials were conducted in a corn wet mill with a 7000-L membrane recycle bioreactor (MRB) that integrated ceramic microfiltration membranes in a semi-closed loop configuration with a stirred-tank reactor . Residence times of 7.5-10 h with ethanol outputs of 10-11.5% (v/v) were obtained when the cell concentration was 60-100 g/L dry wt of yeast, equivalent to about 10(9)-10(10) cells/mL . The performance of the membrane was dependent on the startup mode and pressure management techniques . A steady flux of 70 L/(m2 x h) could be maintained for several days before cleaning was necessary . The benefits of the MRB include better productivity; a clear product stream containing no particulates or yeast cells, which should improve subsequent stripping and distillation operations; and substantially reduced stillage handling . The capital cost of the MRB is $21-$34/(m3 x yr) ($0.08-$0.13/{gal x yr}) of ethanol capacity . Operating cost, including depreciation, energy, membrane replacement, maintenance, labor, and cleaning, is $4.5-9/m3 ($0.017-$0.034/gal) of ethanol.

J Agric Food Chem, 2002 Apr 24, 50(9), 2543 - 7
Continuous whey fermentation using kefir yeast immobilized on delignified cellulosic material; Kourkoutas Y et al.; Delignified cellulosic-supported biocatalyst, prepared by immobilization of kefir yeast on delignified cellulosic material (DCM), was found to be suitable for continuous, modified whey fermentation . The modified whey contained 1% raisin extract and molasses . Ethanol productivities ranged from 3.6 to 8.3 g L(-1)day(-1), whereas parameters such as ethanol concentration, residual sugars, and daily fermented whey productivity were acceptable for the production of potable alcohol and alcoholic drinks in industrial fermentations . The continuous fermentation bioreactor was operated for 39 days, stored for 18 days at 4 degrees C, and operated again for another 15 days without any diminution of the ethanol productivity . The concentrations of higher alcohols (propanol-1, isobutyl alcohol, and amyl alcohols) were low . The main volatile byproducts formed in the continuous process were similar to those observed in alcoholic beverages, and the fermented whey had a good aroma . The concentrations of higher alcohols were very low when compared to that of ethyl acetate, therefore resulting in a quality product . The possibility of using such a process for the production of potable alcohol or a novel, low-alcohol content drink is proposed.

Appl Microbiol Biotechnol, 2002 Apr, 58(5), 645 - 50 Epub 2002 Feb 19.
Construction of a flocculent Saccharomyces cerevisiae strain secreting high levels of Aspergillus niger beta-galactosidase; Domingues L et al.; A flocculent Saccharomyces cerevisiae strain secreting Aspergillus niger beta-galactosidase activity was constructed by transforming S . cerevisiae NCYC869-A3 strain with plasmid pVK1.1 harboring the A . niger beta-galactosidase gene, lacA, under the control of the ADH1 promoter and terminator . Compared to other recombinant S . cerevisiae strains, this recombinant yeast has higher levels of extracellular beta-galactosidase activity . In shake-flask cultures, the beta-galactosidase activity detected in the supernatant was 20 times higher than that obtained with previously constructed strains (Domingues et al . 2000a) . In bioreactor culture, with cheese-whey permeate as substrate, a yield of 878.0 nkat/gsubstrate was obtained . The recombinant strain is an attractive alternative to other fungal beta-galactosidase production systems as the enzyme is produced in a rather pure form . Moreover, the use of flocculating yeast cells allows for enzyme production with high productivity in continuous fermentation systems with facilitated downstream processing.

Curr Opin Biotechnol, 2002 Apr, 13(2), 161 - 6
Plantibodies: applications, advantages and bottlenecks; Stoger E et al.; Various strategies have been developed to exploit plants as bioreactors for the production of pharmaceutical antibodies, to engineer antibody-mediated pathogen resistance or to alter the plant phenotype by immunomodulation . Recent research developments focus on the fine-tuning of expression systems and the detailed characterisation of recombinant products, including the implications of plant-specific glycosylation . Meanwhile, the first of these plant-derived antibody products has successfully completed early phase clinical trials.

Curr Opin Biotechnol, 2002 Apr, 13(2), 124 - 7
Bioprocess monitoring; Harms P et al.; Electrochemical sensors for pH and dissolved oxygen remain the most commonly used in bioprocess monitoring, but continued research has resulted in improved optical sensors . Optical sensors for dissolved oxygen and dissolved carbon dioxide are now commercially available . Advances in optics and electronics are further driving down the costs of these sensors . In the near future, bioprocess optimization will change paradigms as massively parallel, fully instrumented bioreactors become available and high-throughput bioprocessing becomes a reality.

Environ Technol, 2002 Feb, 23(2), 199 - 206
Biodegradation of the organochlorine pesticide, lindane by a sub-tropical white rot fungus in batch and packed bed bioreactor systems; Tekere M et al.; The degradation of the organochlorine pesticide, lindane by a sub-tropical white rot fungus DSPM95 was studied in stationary batch and packed bed bioreactor systems . Pesticide concentrations of 5, 10, 20 and 40 ppm were used in batch cultures while concentrations of 1, 2, 5 and 10 ppm were used in the packed bed bioreactor . Biodegradation of 82 +/- 6% was achieved in batch cultures at concentrations of 5 and 10 ppm . The highest percent degradation achieved in the packed bed reactor was 81% at lindane concentrations of 1 and 2 ppm and the degradation decreased at 10 ppm . The amount of lindane degraded was directly proportional to the initial lindane concentration in the medium . Laccase and manganese peroxidase were produced in both stationary batch and in immobilised cultures, however high enzyme levels could not be sustained for long periods in the packed bed bioreactor.

Biotechnol Bioeng, 2002 May 20, 78(4), 467 - 72
Continuous pH monitoring in a perfused bioreactor system using an optical pH sensor; Jeevarajan AS et al.; Monitoring and regulating the pH of the solution in a bioprocess is one of the key steps in the success of bioreactor operation . An in-line optical pH sensor, based on the optical absorption properties of phenol red present in the medium, was developed and tested in this work for use in NASA space bioreactors based on a rotating wall-perfused vessel system supporting a baby hamster kidney (BHK-21) cell culture . The sensor was tested over three 30-day and one 124-day cell runs . The pH sensor initially was calibrated and then used during the entire cell culture interval . The pH reported by the sensor was compared to that measured by a fiber optically coupled Shimadzu spectrophotometer and a blood gas analyzer . The maximum standard error of prediction for all the four cell runs for development pH sensor against BGA was +/-0.06 pH unit and for the fiber optically coupled Shimadzu spectrophotometer against the blood gas analyzer was +/-0.05 pH unit . The pH sensor system performed well without need of recalibration for 124 days .

Biotechnol Bioeng, 2002 May 20, 78(4), 433 - 41
Control of the redox potential by oxygen limitation improves bacterial leaching of chalcopyrite; Third KA et al.; Shake flask and stirred tank bioleaching experiments showed that the dissolution of chalcopyrite is inhibited by ferric ion concentrations as low as 200 mg L(-1) and redox potentials >420 mV (vs . Ag/AgCl) . Chemical leaching of chalcopyrite (4% suspension, surface area 2.3 m2 g(-1)) was enhanced fourfold in the presence of 0.1 M ferrous sulphate compared with 0.1 M ferric sulphate . A computer-controlled reactor was designed to function as a "potentiostat"-bioreactor by arresting the air supply to the reactor when the redox potential in solution was greater than a designated setpoint . Leaching at a low, constant redox potential (380 mV vs . Ag/AgCl) achieved final copper recoveries of 52%-61%, which was twice that achieved with a continuous supply of oxygen (<30% extraction) . The bacterial populations were observed to continue growing under oxygen limitation but in a controlled manner that was found to improve chalcopyrite dissolution . As the control mechanism is easily established and is likely to decrease production cost, the use of this technology may find application in industry .

Biotechnol Bioeng, 2002 May 20, 78(4), 385 - 94
Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems; Collen A et al.; Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule . The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei . We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations . Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags . The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase . The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch . A recombinant strain of T . reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1 . The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations . Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases . Consequently, only low production levels were obtained in large-scale production trials .

Brain Res Dev Brain Res, 2002 Mar 31, 134(1-2), 103 - 13
Expansion of mammalian neural stem cells in bioreactors: effect of power input and medium viscosity; Sen A et al.; Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cells and progenitor cells . However, it is important to limit the size of the aggregates, as necrotic centers may develop at very large diameters . Previously, we have shown that the hydrodynamics within a suspension bioreactor can be used to control the diameter of NSC aggregates (D(MAVG)<150 microm) below sizes where necrosis would be expected to occur . In the present study, power law correlations were developed for our bioreactors showing the dependence of the maximum mean aggregate diameter on both the kinematic viscosity of the medium and the power input per unit mass of medium . The power input was manipulated by changing the agitation rate (60-100 rpm), and the viscosity was manipulated through the addition of non-toxic levels of carboxymethylcellulose . The study also confirmed that the maximum liquid shear generated at the surface of the aggregates was sufficient to dislodge single cells, thus limiting the maximum diameter of the aggregates, without causing cell damage (tau(max)=9.76 dyn/cm(2)) . This is a first step in the development of a reproducible, scaled-up process for the production of neural stem cells for therapeutic applications including the treatment of neurodegenerative disorders and acute central nervous system injuries.

Waste Manag, 2002, 22(1), 7 - 17
Bioreactor landfills: experimental and field results; Warith M; Bioreactor landfills allow a more active landfill management that recognizes the biological, chemical and physical processes involved in a landfill environment . This paper presents the results of an experimental study carried out to determine the effect of solid waste size, leachate recirculation and nutrient balance on the rate of municipal solid waste (MSW) biodegradation . Higher rates of MSW biodegradation eventually cause a reduction of the contaminant life span of the landfill and decrease in the cost of long term monitoring . The study indicated that the smaller the size of the MSW the faster the biodegradation rate of the waste . In addition, the paper presents the results of leachate recirculation on solid waste biodegradation in a full-scale landfill site, which is located in Nepean, Ontario, Canada . The leachate was recirculated into the landfilled solid waste for 8 years through infiltration lagoons . Similar results to those obtained in the laboratory scale experiments were noted . The average pH of the leachate in the early stages of recirculation was on the acidic range of the pH scale, however, the pH value was in the range of 7-8 after 2 years of leachate recirculation . The concentration of chloride remained fairly constant at about 1000 mg/l during the leachate recirculation period . A decreasing trend of the organic load, measured as biological oxygen demand and chemical oxygen demand, was observed . Recovery of landfill air space was also noted because of the enhanced subsidence and decomposition of the solid waste.

Hepatogastroenterology, 2002 Jan-Feb, 49(43), 91 - 5
Development and perspectives of bioartificial liver support; Naruse K et al.; Bioartificial liver support systems containing adsorbent devices, xenogeneic whole liver perfusion, and hybrid bioartificial liver are anticipated to be effective for the treatment of severe hepatic failure . At present, whole liver perfusion and the hybrid bioartificial liver are two mainstreams in the field of the bioartificial liver, but it is still unclear whether either of them has significant beneficial effects in hepatic failure patients . We developed a new system of xenogeneic direct hemoperfusion consisting of a leukocyte adsorbent, an immunoglobulin adsorbent, and a substitute unit for hepatic function, that is, whole liver or bioreactor . Using this system, the perfusion efficiency rate and oxygen supply of the substitute unit for hepatic function were significantly increased . A further improvement of our system by incorporating a leukocytapheresis device to return the leukocyte-rich portion of the perfused blood back to the patient's body directly was undergone . Moreover, our system can be applied not only as a bioartificial liver, but also as a bioartificial kidney . To solve the problem of xenogeneic protein influx into the human body associated with extracorporeal bioartificial liver support, it is important to develop transgenic cattle in which human protein genes are transduced . Influx of porcine endogenous retrovirus into the human body has been a controversial subject . In relation to this issue, the cross-hemoperfusion method is promising in that the patient's circuit and the bioartificial liver circuit are separated by a semipermeable membrane, which can prevent any kind of virus from flowing into the patient's circuit.

Biotechnol Prog, 2002 Mar-Apr, 18(2), 337 - 45
Passaging protocols for mammalian neural stem cells in suspension bioreactors; Sen A et al.; Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders . As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC . Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media . The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time . We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter . When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur . Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80% . The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates . We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.

Int J Food Microbiol, 2002 Mar, 73(2-3), 145 - 57
Optimal temperature input design for estimation of the square root model parameters: parameter accuracy and model validity restrictions; Bernaerts K et al.; As part of the model building process, parameter estimation is of great importance in view of accurate prediction making . Confidence limits on the predicted model output are largely determined by the parameter estimation accuracy that is reflected by its parameter estimation covariance matrix . In view of the accurate estimation of the Square Root model parameters, Bernaerts et al . have successfully applied the techniques of optimal experiment design for parameter estimation {Int . J . Food Microbiol . 54 (1-2) (2000) 27} . Simulation-based results have proved that dynamic (i.e., time-varying) temperature conditions characterised by a large abrupt temperature increase yield highly informative cell density data enabling precise estimation of the Square Root model parameters . In this study, it is shown by bioreactor experiments with detailed and precise sampling that extreme temperature shifts disturb the exponential growth of Escherichia coli K12 . A too large shift results in an intermediate lag phase . Because common growth models lack the ability to model this intermediate lag phase, temperature conditions should be designed such that exponential growth persist even though the temperature may be changing . The current publication presents (i) the design of an optimal temperature input guaranteeing model validity yet yielding accurate Square Root model parameters, and (ii) the experimental implementation of the optimal input in a computer-controlled bioreactor . Starting values for the experiment design are generated by a traditional two-step procedure based on static experiments . Opposed to the single step temperature profile, the novel temperature input comprises a sequence of smaller temperature increments . The structural development of the temperature input is extensively explained . High quality data of E . coli K12 under optimally varying temperature conditions realised in a computer-controlled bioreactor yield accurate estimates for the Square Root model parameters . The latter is illustrated by means of the individual confidence intervals and the joint confidence region.

Appl Phys B, 2002 Jan, 74(1), 85 - 93
Applications of Kalman filtering to real-time trace gas concentration measurements; Leleux DP et al.; A Kalman filtering technique is applied to the simultaneous detection of NH3 and CO2 with a diode-laser-based sensor operating at 1.53 micrometers . This technique is developed for improving the sensitivity and precision of trace gas concentration levels based on direct overtone laser absorption spectroscopy in the presence of various sensor noise sources . Filter performance is demonstrated to be adaptive to real-time noise and data statistics . Additionally, filter operation is successfully performed with dynamic ranges differing by three orders of magnitude . Details of Kalman filter theory applied to the acquired spectroscopic data are discussed . The effectiveness of this technique is evaluated by performing NH3 and CO2 concentration measurements and utilizing it to monitor varying ammonia and carbon dioxide levels in a bioreactor for water reprocessing, located at the NASA-Johnson Space Center . Results indicate a sensitivity enhancement of six times, in terms of improved minimum detectable absorption by the gas sensor.

In Vitro Cell Dev Biol Anim, 2002 Feb, 38(2), 118 - 22
Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity; Sundaresan A et al.; Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response . Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen . We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments . Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells {PBMCs}) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected . In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity . Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87% . Augmentation of cellular calcium flux with ionomycin had no restorative effect . Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion . Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures . Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.

Nat Biotechnol, 2002 Apr, 20(4), 396 - 9
Expression of exogenous protein in the egg white of transgenic chickens; Harvey AJ et al.; Using a replication-deficient retroviral vector based on the avian leukosis virus (ALV), we inserted into the chicken genome a transgene encoding a secreted protein, beta-lactamase, under the control of the ubiquitous cytomegalovirus (CMV) promoter . Biologically active beta-lactamase was secreted into the serum and egg white of four generations of transgenic chickens . The expression levels were similar in successive generations, and expression levels in the magnum of the oviduct were constant over at least 16 months in transgenic hens, indicating that the transgene was stable and not subject to silencing . These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active proteins in egg white.

Biotechnol Bioeng, 2002 May 5, 78(3), 321 - 32
A kinetic model describing cell growth and production of highly active, recombinant ice nucleation protein in Escherichia coli; Palaiomylitou MA et al.; A structured kinetic model, which describes the production of the recombinant ice nucleation protein in different conditions, was applied . The model parameters were estimated based on the variation of the specific growth rate and the intracellular product concentration during cultivation . The equations employed relate the cellular plasmid content or plasmid copy number with the cloned-gene expression; these correlations were successfully tested on the experimental data . The optimal nutrient conditions for the growth of Escherichia coli expressing the inaZ gene of Pseudomonas syringae were determined for the production of active ice nucleation protein . The kinetics of the cultures expressing the inaZ gene were studied in a bioreactor at different growth temperatures and nutrient conditions .

Biotechnol Bioeng, 2002 May 5, 78(3), 257 - 69
A microfabricated array bioreactor for perfused 3D liver culture; Powers MJ et al.; We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy . Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls . Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel . Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses . We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period . We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability . We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy .

J Bone Miner Res, 2002 Apr, 17(4), 652 - 60
Cartilage calcification studied by proton nuclear magnetic resonance microscopy; Potter K et al.; A three-dimensional (3D) mineralizing culture system using hollow fiber bioreactors has been developed to study the early stages of endochondral ossification by proton nuclear magnetic resonance (NMR) microscopy . Chondrocytes harvested from the cephalic half of the sterna from 17-day-old chick embryos were terminally differentiated with 33 nM of retinoic acid for 1 week and mineralization was initiated by the addition of 1% beta-glycerophosphate to the culture medium . Histological sections taken after 6 weeks of development in culture confirmed calcification of the cartilage matrix formed in bioreactors . Calcium to phosphorus ratios (1.62-1.68) from X-ray microanalysis supported electron diffraction of thin tissue sections showing the presence of a poorly crystalline hydroxyapatite mineral phase in the cultures . After 4 weeks of culture, quantitative proton NMR images showed water proton magnetization transfer rate constants (km) were higher in premineralized cartilage compared with uncalcified cartilage, a result suggesting collagen enrichment of the matrix . Notably after 5 weeks mineral deposits formed in bioreactors principally in the collagen-enriched zones of the cartilage with increased km values . This caused marked reductions in water proton longitudinal (T1) and transverse (T2) relaxation times and water diffusion coefficients (D) . These results support the hypothesis that mineralization proceeds in association with a collagen template . After 6 weeks of culture development, the water proton T2 values decreased by 13% and D increased by 7% in uncalcified areas, compared with the same regions of tissue examined 1 week earlier . These changes could be attributed to the formation of small mineral inclusions in the cartilage, possibly mediated by matrix vesicles, which may play an important role in cartilage calcification . In summary, NMR images acquired before and after the onset of mineralization of the same tissue provide unique insights into the matrix events leading to endochondral mineral formation.

J Biotechnol, 2001 Dec, 82(2), 123 - 35
Sampling and monitoring in bioprocessing using microtechniques; Gastrock G et al.; In this review we describe aspects of interactions between bioreactors and analytical systems including microsystems . Principles of bioprocess monitoring are summarized, before we focus on the miniaturization of sampling systems guaranteeing bioprocess sterility and providing analytical systems with a liquid sample . The application of negative dielectrophoresis as a new principle for cell retention in a sampling system is described followed by theoretical aspects and results . Properties of micromachined silicon membranes as filters for sampling systems and for biosensor protection are discussed.

Trends Biotechnol, 2002 Apr, 20(4), 135 - 7
Can imperfections help to improve bioreactor performance?
Patnaik PR.
Pilot-scale and larger bioreactors differ from small laboratory-scale reactors in terms of a greater occurrence of noise and incomplete mixing of the broth . Conventional control tries to induce good mixing and to filter out the noise as completely as possible . As such an 'ideal' operation is difficult to achieve, recent work has tried to exploit the non-ideal features to improve the performance . Using artificial neural networks, the degree of mixing, the extent of filtering of noise and the distribution of plasmid copy number (in a recombinant fermentation) can be controlled effectively on-line . This strategy generates better productivities than well-mixed noise-free operations, which suggests that deviations from ideal behaviour should be gainfully harnessed and not suppressed.

Int J Artif Organs, 2002 Feb, 25(2), 151 - 7
Combined effect of oxygen and ammonia on the kinetics of ammonia elimination and oxygen consumption of adherent rat liver cells; Catapano G et al.; Oxygen is essential for the survival of isolated liver cells and its concentration is known to affect their viability and function . Recent reports have also shown that ammonia is eliminated at a rate depending on its concentration and that high ammonia concentrations may be cytotoxic to rat liver cells . Nonetheless, little quantitative information on the effect of either metabolite on liver cell reaction kinetics is available although important to the design of bioreactors for bioartificial livers (BALs) . In this investigation, we characterized the dependence of the rate of oxygen consumption (OCR), ammonia elimination (AER) and urea synthesis (USR) on ammonia concentration at physiological (i.e., 43 and 72 mmHg) and supra-physiological (i.e., 134 mmHg) dissolved oxygen tensions . To this purpose, isolated rat liver cells were cultured in adhesion on collagen in a continuous-flow bioreactor optimised for the kinetic characterisation of liver cell metabolic reactions . Rates of the investigated reactions generally increased with increasing ammonia concentrations . OCR and USR significantly increased with increasing dissolved oxygen tensions, particularly at high ammonia concentrations . The actual dissolved oxygen tension significantly influenced also OCR and USR dependence on ammonia concentration . The best-fit rate equations were used to show that, at the beginning of the treatment with a bioreactor packed with primary liver cells, high ammonia concentration in the blood may cause large hypoxic zones in the bioreactor as a result of its effect on OCR . This suggests that plasma (or blood) detoxification prior to entering the bioreactor might enhance BAL efficacy by preserving a large fraction of the available cell activity for longer times.

Water Sci Technol, 2002, 45(3), 11 - 24
The sewer as a bioreactor--a dry weather approach; Hvitved-Jacobsen T et al.; The sewer is a reactor for chemical and microbial transformations of wastewater . These in-sewer processes affect the quality of the wastewater and thereby the sewer itself, the subsequent treatment and the receiving water quality . The paper focuses on the interactions between the dry weather in-sewer chemical and microbial transformations of the wastewater and the corresponding processes in a downstream located treatment plant . A conceptual understanding of the sewer processes is crucial in this respect.

Ann Transplant, 2001, 6(3), 40 - 6
Review of a flat membrane bioreactor as a bioartificial liver; De Bartolo L et al.; Recent developments in tissue engineering permit to use isolated hepatocytes in a bioreactor for the creation of a bioartificial liver which supports patients suffering from acute liver failure . In this study, the authors discuss the development of a flat membrane bioreactor using pig hepatocytes for the replacement of liver functions . The flat membrane bioreactor permits a high-density hepatocyte culture under sufficient oxygenation conditions, comparable to an in vivo microenvironment . In this bioreactor, built according to the in vivo organisation of the liver, pig hepatocytes are cultured with non-parenchymal cells within an extracellular matrix between oxygen-permeable flat-sheet membranes as individual plates . The performance of the "scale-up bioreactor" was tested in vitro for 18 days in static and flux conditions . Pig hepatocytes in the bioreactor were maintained in three-dimensional co-culture with non-parenchymal cells and are reorganised in a way similar to the liver cell plates in vivo: cells remained polarised in vitro clearly demonstrating biliary zones surrounding individual hepatocytes . The biochemical performance of the bioreactor was assessed by estimating its ability to remove two of the major toxins associated with hepatic encephalopathy: benzodiazepines and ammonia . The rates of ammonia elimination and drug biotransformation were maintained at constant high levels for almost two weeks . This "scaled-up bioreactor" provides conditions favourable for the formation of contiguous cell sheets, which allow to maintain constant liver specific functions.

J Hazard Mater, 2002 Mar 29, 90(3), 311 - 21
Brewery wastewater treatment in a fluidised bed bioreactor; Ochieng A et al.; A hydrodynamic characteristic performance of a three phase fluidised bed bioreactor has been studied with brewery wastewater . The influence of operating parameters, such as phase hold up, phase mixing, aspect ratio and superficial gas velocity, on an aerobic biodegradation in a bioreactor of 0.16 m i.d . and 2.7m in height, was analysed . A low-density (960 kg/m(3)) support particle with an internal interstice was employed . The particle and liquid loading were varied in order to determine the effect of phase hold up on bed homogeneity . The ranges in which particle loading and bed height affect fluidisation, and consequently chemical oxygen demand (COD) reduction, were determined . The distributor used in this work was designed such that fluid flow pattern similar to that of a draft tube was induced in the reactor . The low-density particles enabled cost effective operation at a relatively low gas superficial velocity (2.5 cm/s) . Aspect ratio significantly influenced the overall bed homogeneity, and the optimum aspect ratio was 10, with volume of the support particles being 21% of the reactor volume.

J Cell Biochem, 2002, 85(1), 167 - 79
Characterization of the osteoblast-like cell phenotype under microgravity conditions in the NASA-approved Rotating Wall Vessel bioreactor (RWV); Rucci N et al.; Weightlessness induces bone loss in humans and animal models . We employed the NASA-approved Rotating Wall Vessel bioreactor (RWV) to develop osteoblast-like cell cultures under microgravity and evaluate osteoblast phenotype and cell function . Rat osteoblast-like cell line (ROS.SMER#14) was grown in the RWV at a calculated gravity of 0.008g . For comparison, aliquots of cells were grown in conventional tissue culture dishes or in Non-Rotating Wall Vessels (N-RWV) maintained at unit gravity . In RWV, osteoblasts showed high levels of alkaline phosphatase expression and activity, and elevated expression of osteopontin, osteocalcin, and bone morphogenetic protein 4 (BMP-4) . In contrast, the expression of osteonectin, bone sialoprotein II and BMP-2 were unaltered compared to cells in conventional culture conditions . These observations are consistent with a marked osteoblast phenotype . However, we observed that in RWV osteoblasts showed reduced proliferation . Furthermore, DNA nucleosome-size fragmentation was revealed both morphologically, by in situ staining with the Thymine-Adenine binding dye bis-benzimide, and electrophoretically, by DNA laddering . Surprisingly, no p53, nor bcl-2/bax, nor caspase 8 pathways were activated by microgravity, therefore the intracellular cascade leading to programmed cell death remains to be elucidated . Finally, consistent with an osteoclast-stimulating effect by microgravity, osteoblasts cultured in RWV showed upregulation of interleukin-6 (IL-6) mRNA, and IL-6 proved to be active at stimulating osteoclast formation and resorbing activity in vitro . We conclude that under microgravity, reduced osteoblast life span and enhanced IL-6 expression may result in inefficient osteoblast- and increased osteoclast-activity, respectively, thus potentially contributing to bone loss in individuals subjected to weightlessness.

Transfus Clin Biol, 2002 Jan, 9(1), 9 - 14
In vivo studies of monoclonal anti-D and the mechanism of immune suppression; Kumpel BM; Administration of anti-D immunoglobulin to D- women after delivery of a D+ infant has dramatically reduced the number of immunised women and cases of haemolytic disease of the fetus and newborn . The use of monoclonal anti-D might alleviate some of the pressures on maintaining adequate supplies of plasma sourced anti-D . Two human monoclonal antibodies, BRAD-3 (IgG1) and BRAD-5 (IgG3), with proven activity in in vitro functional (immunological) assays with cells bearing IgG Fc receptors (Fc gamma R) were selected for clinical studies . They were prepared by purification of IgG secreted by culture of the Epstein-Barr virus-transformed B cell lines in hollow fibre bioreactors . The clearance of D+ red cells injected into D- subjects was accelerated by prior injection of the monoclonal antibodies, both individually and blended (3:1, BRAD-5: BRAD-3) . The subjects were protected from Rh D immunisation . A large multicentre study evaluated the BRAD-3/5 blend for its ability to prevent Rh D immunisation in 95 D- subjects given 400 micrograms i.m . 24 hours after injection of 5 ml D+ red cells . Challenge injections of D+ red cells alone were given 24 and 36 weeks later, and blood samples were taken every 4 weeks from the subjects throughout the study for detection of anti-D responses . There was one definite and one possible failure of protection; in one subject the plasma anti-D level rose from week 12 onwards, and in another individual rapid seroconversion was observed at week 28 . Considering the relatively large dose of red cells and the number of subjects studied, it was concluded that the failure rate was much lower than in routine Rh D prophylaxis . The responder rate was 13% by week 36 and 24% by week 48 . The low percentage of responders and the modest levels of endogenous anti-D produced suggested that administration of monoclonal anti-D had induced long-term specific suppression of anti-D responses in these subjects . The most likely mechanism of action was considered to be inhibition of B cells resulting from co-crosslinking antigen receptors with inhibitory Fc gamma R when the B cells contacted red cells that had bound passive anti-D.

J Cardiovasc Surg (Torino), 2002 Apr, 43(2), 275 - 9
Tissue engineered tracheal prosthesis with acceleratedly cultured homologous chondrocytes as an alternative of tracheal reconstruction; Lee CJ et al.; BACKGROUND: Autologous tissue is an ideal substitue for an extensive tracheal reconstruction, but it is rarely feasible in clinical situations . Many tracheal prosthesis had been used for such an instances, but unfortunately it is still problematic . Dislocation, local infection, hemorrage, and luminal stenosis can cause prosthetic failure . To achieve clinically available autologous tracheal prosthesis, it is necessary that we have to get phenotypically functioning chondrocytes, rapid differentiation of harvested autologous chondrocytes, and the survival of free grafted cultured chondrocytes . METHODS: In this study, we investigated isolation and culture method of the chondrocytes using the rabbit costal cartilage, and the cells were characterized microscopically and biochemically first . Then we have used cultured rabbit chondrocytes to investigate the role of growth factors upon the proliferation and regulation of the cultured chondrocytes . We have examined the effect of peptide growth factors on DNA and proteoglycan synthesis to the rabbit chondrocyte . The effects of IGF-I and basic FGF were investigated individually . Secondly, acceleratedly cultured chondrocytes were embeded to polymer (PLGA) scaffold in bioreactor, and implanted to defected rabbit trachea . Six weeks later, the rabbits were sacrificed and examined their histologic characteristics . RESULTS: The harvested chondrocytes from costal arch grew well and were amplified successfully maitaining their own phenotypes . Its embedding to PLGA scaffold was accomplished successfully . The implanted tracheal prosthesis maintains its physical integrity well, but the histologic examination revealed non-viable chondrocytes . The epithelial linings were good . CONCLUSIONS: The tissue engineered tracheal prosthesis can be a promising alternative of good functional air way tube in short term experiment, but biologically not vital yet . Further investigations are necessary to see the survival of free grafted chondrocytes and the long term results.

Transplantation, 2002 Feb 15, 73(3), 420 - 9
Clinical and laboratory evaluation of the safety of a bioartificial liver assist device for potential transmission of porcine endogenous retrovirus; Kuddus R et al.; BACKGROUND: The potential risk of transmission of porcine endogenous retroviruses (PERV) from xenogeneic donors into humans has been widely debated . Because we were involved in a phase I/II clinical trial using a bioartificial liver support system (BLSS), we proceeded to evaluate the biosafety of this device . MATERIALS AND METHODS: The system being evaluated contains primary porcine hepatocytes freshly isolated from pathogen-free, purpose-raised herd . Isolated hepatocytes were installed in the shell, which is separated by a semipermeable membrane (100-kD nominal cutoff) from the lumen through which the patients' whole blood is circulated . Both before and at defined intervals posthemoperfusion, patients' blood was obtained for screening . Additionally, effluent collected from a clinical bioreactor was analyzed . The presence of viral particles was estimated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RT assays . For the detection of pig genomic and mitochondrial DNA, sequence-specific PCR (SS-PCR) was used . Finally, the presence of infectious viral particles in the samples was ascertained by exposure to the PERV-susceptible human cell line HEK-293 . RESULTS: PERV transcripts, RT activity, and infectious PERV particles were not detected in the luminal effluent of a bioreactor . Culture supernatant from untreated control or mitogen-treated porcine hepatocytes (cleared of cellular debris) also failed to infect HEK-293 cell lines . Finally, RT-PCR, SS-PCR, and PERV-specific RT assay detected no PERV infection in the blood samples obtained from five study patients both before and at various times post-hemoperfusion . CONCLUSION: Although longer patient follow-up is required and mandated to unequivocally establish the biosafety of this device and related bioartificial organ systems, these analyses support the conclusion that when used under standard operational conditions, the BLSS is safe.

Indian J Physiol Pharmacol, 2001 Jul, 45(3), 367 - 72
Effect of addition of proton carriers in culture medium on growth and secretion of hybridoma cell line OKT3; Datta D et al.; Monoclonal antibodies (MAb) constitute the centre of all in-vitro diagnostic measures and almost all in-vivo therapeutic manoeuvres now . Production emphasis for these antibodies is having a current shift from animal-based large-scale culture to in-vitro bioreactor-based high-density culture . One of the major difficulties in high-density culture is end-metabolite accumulation in batch and fed-batch cultures in the forms of H+, NH4+ etc. . thereby reducing cellular growth and secretions . In the present study, effects of added proton carries--NAD and NADP--over and above the metabolic pools of the molecules, were examined on the cellular growth and secretion kinetics . Although NADP fortification showed a remarkable improvement in cellular growth (time dependent 200-300% improvements compared to controls) and size, cumulative MAb titre was better with NAD fortification . Combined additional loads of the proton carriers would be interesting to study in high density culture conditions.

J Biotechnol, 2002 Apr 25, 95(1), 39 - 48
Measurement of hydrophobic interactions of mammalian cells grown in culture; Ghebeh H et al.; An assay was developed to measure the hydrophobic interactions of commonly used mammalian cell lines grown in culture . The assay depends on the loss of cells from an aqueous suspension following vortexing with a hydrophobic oil phase . This allowed the determination of a hydrophobicity index, which was significantly higher for Chinese Hamster Ovary (CHO) cells than either a murine hybridoma (CC9C10) or a myeloma (SP2/0) . This suggests that CHO cells may have a higher intrinsic cell surface hydrophobicity . The assay was also used to study the effect of different additives on the hydrophobic interactions of the cells . A dose-dependent effect was shown for the non-ionic surfactant, Pluronic F68, in reducing the hydrophobic interaction of the CHO cells . However, the pattern of the decrease due to Pluronic F68 was different for each cell line . A higher concentration of Pluronic F68 (0.2%) was required to eliminate the hydrophobic interactions of CHO cells compared to either myelomas or hybridomas, where only 0.05% was required to reduce these interactions to a similar level . Several oils were found suitable for this assay although canola oil maximized the sensitivity of the measured changes . The assay may be useful in monitoring changes in the hydrophobic interactions of mammalian cells during growth in bioreactors . This may be important in optimizing the concentration of cell protectants such as Pluronic F68 in agitated cultures.

J Biotechnol, 2002 Apr 25, 95(1), 13 - 23
Effect of carbon source and aeration rate on broth rheology and fungal morphology during red pigment production by Paecilomyces sinclairii in a batch bioreactor; Cho YJ et al.; The influence of carbon source and aeration rate on fermentation broth rheology, mycelial morphology and red pigment production of Paecilomyces sinclairii was investigated in a 5-l stirred-tank bioreactor . The characteristics of P . sinclairii grown on starch and on sucrose medium were comparatively studied: the specific growth rate in sucrose medium (0.04 h(-1)) was higher than that in starch medium, whereas the specific production rate of red pigments (0.04 gg(-1)d(-1)) was favorable in starch medium . P . sinclairii grown in sucrose medium were highly branched and showed longer hyphal lengths than that in starch medium . The consistency index (K) in sucrose medium was markedly higher than that in starch medium due to higher cell mass, while the higher values of flow behavior index (n) were indicated at the late stationary phase in starch medium . The aeration rate was varied within the ranges from 0.5 to 3.5 vvm while running the fermentation at mild agitation of 150 rpm using sucrose as the carbon source . The maximum biomass concentration of P . sinclairii was about 33 gl(-1) with an aeration rate of 1.5 vvm, whereas the maximum yield of red pigment production (4.73 gl(-1)) was achieved with 3.5 vvm . The highly branched cell morphology appeared at 1.5 vvm and the highly vacuolated cell morphology was observed in a high aeration rate (3.5 vvm) . There was no significant variance in rheological parameters (K and n) between culture broths from different aeration conditions.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 255 - 9
Pure bacterial isolates that convert p-xylene to terephthalic acid; Bramucci MG et al.; Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds . Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 170 - 4
Production and biotransformation of 6-pentyl-alpha-pyrone by Trichoderma harzianum in two-phase culture systems; Serrano-Carreon L et al.; The final concentration of 6-pentyl-a-pyrone (6PP) produced in cultures of Trichoderma spp . is limited by the fact that inhibition of biomass growth occurs at 6PP concentrations as low as 100 mg/l . The aim of this work was to evaluate liquid-liquid extractive fermentation systems as an alternative to overcome the toxicity problems and to increase the production of 6PP by this fungus . Two alkanes (n-decane and n-hexadecane) and two dicarboxylic esters (dibutyl phthalate and dioctyl phthalate) were evaluated in shake flask cultures . The highest 6PP production (173 ppm) was achieved when n-hexadecane was used, being 3.5-fold the maximum 6PP concentration of a culture without the solvent . Cultivation of Trichoderma harzianum in a 10-1 bioreactor with n-hexadecane yielded 6PP production ninefold higher than that from control cultures . However, 6PP production in the bioreactor (83 ppm) was lower than in shake flasks . Differences in the power drawn to the fluid at each scale could account for such behavior . Even in the presence of the solvent, 6PP content decreased after reaching its maximal concentration.

Appl Environ Microbiol, 2002 Mar, 68(3), 1464 - 7
Continuous steady-state method using tenax for delivering tetrachloroethene to chloro-respiring bacteria; Brennan RA et al.; Tenax-TA, a solid-phase sorbent, was used as an alternative to hexadecane for continuous delivery of tetrachloroethene (PCE) to Desulfuromonas strain BB1, a chloro-respiring microorganism . In both batch and bioreactor configurations, Tenax not only maintained low, steady-state concentrations of PCE in an active culture for several months but also adsorbed the product of dechlorination, cis-1,2-dichloroethene, before it approached toxic levels.

Life Support Biosph Sci, 1998, 5(4), 375 - 88
Azolla-Anabaena symbionts and microbial mat as nitrogen-fixing biocatalysts for bioregenerative space life support; Cuello JL et al.; This study was conducted to characterize the responses of selected nitrogen-fixing biocatalysts to various environmental parameters that are likely to be encountered in a scaled-up nitrogen-fixing bioregenerative unit for use in a biologically based life support system (BLSS) . The results showed that while both Azolla filiculoides and Azolla nilotica thrived on standard nitrogen-free liquid media, only Azolla filiculoides maintained its growth rate in the presence of 50% by volume of aerobic-bioreactor potato effluent (ABPE) in its liquid medium . The growth rate and Anabaena cavity population of Azolla filiculoides also remained undiminished in the presence of as much as 10 mM of NO3- in its liquid medium . The tolerance of Azolla filiculoides for NO3- however, lay somewhere between 10 and 20 mM of NO3- . Azolla filiculoides failed to grow at 5 mM or greater concentrations of NH4+ . The growth rate of the Oscillatoria-dominated microbial mat was significantly elevated by increased silage loading density (16 g/L) as well as by adding 5 mM of NO3- into its liquid medium . Neither the growth rate of Azolla filiculoides nor that of the microbial mat was affected by three levels of photosynthetic photon flux (PPF) (520, 265, and 125 micromoles m-2 s-1) under high-pressure sodium or cool fluorescent lamps.

Gravit Space Biol Bull, 2001 Jun, 14(2), 55 - 64
Clinostats and bioreactors; Klaus DM; The environment created on Earth within a clinostat or Rotating Wall Vessel (RWV) bioreactor is often referred to as "simulated microgravity" . Both devices utilize constant reorientation to effectively nullify cumulative sedimentation of particles . Neither, however, can fully reproduce the concurrent lack of structural deformation, displacement of intercellular components and/or reduced mass transfer in the extracellular fluid that occur in actual weightlessness . Parameters including density, viscosity, and even container geometry must each be considered to determine the overall gravity-dependent effects produced by either a clinostat or the RWV bioreactor; in addition, the intended application of these two devices differs considerably . A state of particle "motionlessness" relative to the surrounding bulk fluid, which is nearly analogous to the extracellular environment encountered under weightless conditions, can theoretically be achieved through clinorotation . The RWV bioreactor, on the other hand, while similarly maintaining cells in suspension as they continually "fall" through the medium under 1 g conditions, can also purposefully induce a perfusion of nutrients to and waste from the culture . A clinostat, therefore, is typically used in an attempt to reproduce the quiescent, unstirred fluid conditions achievable on orbit; while the RWV bioreactor ideally creates a low shear, but necessarily mixed, fluid environment that is optimized for suspension culture and tissue growth . Other techniques for exploring altered inertial environments, such as freefall, neutral buoyancy and electromagnetic levitation, can also provide unique insight into how gravity affects biological systems . Ultimately, all underlying biophysical principles thought to give rise to gravity-dependent physiological responses must be identified and thoroughly examined in order to accurately interpret data from flight experiments or ground-based microgravity analogs.

Acta Astronaut, 2001 Mar-Jun, 48(5-12), 287 - 97
Aquatic modules for bioregenerative life support systems based on the C.E.B.A.S . biotechnology {correction of biotechnilogy}; Bluem V et al.; Most concepts for bioregenerative life support systems are based on edible higher land plants which create some problems with growth and seed generation under space conditions . Animal protein production is mostly neglected because of the tremendous waste management problems with tetrapods under reduced weightlessness . Therefore, the "Closed Equilibrated Biological Aquatic System" (C.E.B.A.S.) was developed which represents an artificial aquatic ecosystem containing aquatic organisms which are adapted at all to "near weightlessness conditions" (fishes Xiphophorus helleri, water snails Biomphalaria glabrata, ammonia oxidizing bacteria and the rootless non-gravitropic edible water plant Ceratophyllum demersum) . Basically the C.E.B.A.S . consists of 4 subsystems: a ZOOLOGICAL (correction of ZOOLOGICASL) COMPONENT (animal aquarium), a BOTANICAL COMPONENT (aquatic plant bioreactor), a MICROBIAL COMPONENT (bacteria filter) and an ELECTRONICAL COMPONENT (data acquisition and control unit) . Superficially, the function principle appears simple: the plants convert light energy into chemical energy via photosynthesis thus producing biomass and oxygen . The animals and microorganisms use the oxygen for respiration and produce the carbon dioxide which is essential for plant photosynthesis . The ammonia ions excreted by the animals are converted by the bacteria to nitrite and then to nitrate ions which serve as a nitrogen source for the plants . Other essential ions derive from biological degradation of animal waste products and dead organic matter . The C.E.B.A.S . exists in 2 basic versions: the original C.E.B.A.S . with a volume of 150 liters and a self-sustaining standing time of more than 13 month and the so-called C.E.B.A.S . MINI MODULE with a volume of about 8.5 liters . In the latter there is no closed food loop by reasons of available space so that animal food has to be provided via an automated feeder . This device was flown already successfully on the STS-89 and STS-90 spaceshuttle missions and the working hypothesis was verified that aquatic organisms are nearly not affected at all by space conditions, i.e . that the plants exhibited biomass production rates identical to the sound controls and that as well the reproductive, and the immune system as the embryonic and ontogenic development of the animals remained undisturbed . Currently the C.E.B.A.S . MINI MODLULE is prepared for a third spaceshuttle flight (STS-107) in spring 2001 . Based on the results of the space experiments a series of prototypes of aquatic food production modules for the implementation into BLSS were developed . This paper describes the scientific disposition of the STS-107 experiment and of open and closed aquaculture systems based on another aquatic plant species, the Lemnacean Wolffia arrhiza which is cultured as a vegetable in Southeastern Asia . This plant can be grown in suspension culture and several special bioreactors were developed for this purpose . W . arrhiza reproduces mainly vegetatively by buds but also sexually from time to time and is therefore especially suitable for genetic engineering, too . Therefore it was used, in addition, to optimize the C.E.B.A.S . MINI MODULE to allow experiments with a duration of 4 month in the International Space Station the basic principle of which will be explained . In the context of aquaculture systems for BLSS the continuous replacement of removed fish biomass is an essential demand . Although fish reproduction seems not to be affected in the shortterm space experiments with the C.E.B.A.S . MINI MODULE a functional and reliable hatchery for the production of siblings under reduced weightlessness is connected with some serious problems . Therefore an automated "reproduction module" for the herbivorous fish Tilapia rendalli was developed as a laboratory prototype . It is concluded that aquatic modules of different degrees of complexity can optimize the productivity of BLSS based on higher land plants and that they offer an unique opportunity for the production of animal protein in lunar or planetary bases . c2001 Elsevier Science Ltd . All rights reserved.

Biotechnol Bioeng, 2002 Apr 5, 78(1), 73 - 80
Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress; Camisard V et al.; A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress . The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus . This technique allows the acquisition of microscopic still images without mechanical sampling techniques . The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor . The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used) . Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s . Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM . The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer . The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor . Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H . anomala . The volume signal of H . anomala during osmotic shocks showed a very fast cell contraction within less than a second . Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration . These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures .

Huan Jing Ke Xue, 2001 Nov, 22(6), 99 - 102
{Bioreactor-landfill site for leaching solution treatment}; He R et al.; The study utilizes the system combined bioreactor with landfill site to treat leachate . The results show that the system helped the degradation of the organic pollutants in the leachate to be divided into two-phases, the hydrolytic fermentation and acid-production phases mainly occurred in the landfill site, the acidification rate was 40%-50%, the methane-production chiefly occurred in the bioreactor(UASB) . It treated the leachate significantly and benefits the collection and utilization of methane gas . Also, the system accelerates the process of degrading municipal solid waste and stabilizing landfill site.

World J Gastroenterol, 2001 Dec, 7(6), 826 - 9
TECA hybrid artificial liver support system in treatment of acute liver failure; Xue YL et al.; AIM: To assess the efficacy and safety of TECA type hybrid artificial liver support system (TECA-HALSS) in providing liver function of detoxification, metabolism and physiology by treating the patients with acute liver failure (ALF) . METHODS: The porcine liver cells (1-2) x 10(10) were separated from the Chinese small swine and cultured in the bioreactor of TECA-BALSS at 37.0 degrees C and circulated through the outer space of the hollow fiber tubes in BALSS . The six liver failure patients with various degree of hepatic coma were treated by TECA-HALSS and with conventional medicines . The venous plasma of the patients was separated by a plasma separator and treated by charcoal adsorbent or plasma exchange . The plasma circulated through the inner space of the hollow fiber tubes of BALSS and mixed with the patients' blood cells and flew back to their blood circulation . Some small molecular weight substances were exchanged between the plasma and porcine liver cells . Each treatment lasted 6.0-7.0 h . Physiological and biochemical parameters were measured before,during and after the treatment . RESULTS: The average of porcine liver cells was (1.0-3.0) x 10(10) obtained from each swine liver using our modified enzymatic digestion method . The survival rate of the cells was 85%-93% by trypan blue stain and AO/PI fluorescent stain . After cultured in TECA-BALSS bioreactor for 6 h, the survival rate of cells still remained 70%-85% . At the end of TECA-HALSS treatment, the levels of plasma NH(3), ALT, TB and DB were significantly decreased . The patients who were in the state of drowsiness or coma before the treatment improved their appetite significantly and regained consciousness, some patients resumed light physical work on a short period after the treatment.One to two days after the treatment, the ratio of PTA increased warkedly . During the treatment, the heart rates, blood pressure, respiration condition and serum electrolytes (K(+), Na(+) and Cl(-)) were stable without thrombosis and bleeding in all the six patients . CONCLUSION: TECA-HALSS treatment could be a rapid, safe and efficacious method to provide temporary liver support for patients with ALF.

J Agric Food Chem, 2002 Feb 27, 50(5), 1073 - 6
Conversion of dextran to cycloisomaltooligosaccharides using an enzyme-immobilized porous hollow-fiber membrane; Kawai T et al.; This paper describes a cycloisomaltooligosaccharide glucanotransferase (CITase)-multilayer-immobilized porous hollow-fiber membrane used as an enzyme bioreactor . Dextran, a substrate with an average molecular mass of 43000, is converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides, effective as a preventive for dental caries, aided by convective transport of the substrate to the vicinity of the enzyme through the pores . Epoxy-group-containing graft chains were uniformly appended onto the surface of pores throughout a porous hollow-fiber membrane by radiation-induced graft polymerization . Subsequently, a diethylamino group was introduced, as an anion-exchange moiety, to the graft chains, which caused the chains to expand toward the interior of the pores due to mutual electrostatic repulsion . The expanding graft chain provided multilayer binding sites for CITase . Fifty-five milligrams of adsorbed CITase per gram of membrane is equivalent to the degree of multilayer binding of 5 . Finally, 80% of the multilayer-adsorbed CITase was immobilized via enzymatic cross-linking with transglutaminase to prevent the leakage of enzymes . CITase, with a degree of multilayer immobilization of 4, produced the target cycloisomaltooligosaccharides at a conversion yield of 55% in weight at 310 K during permeation by the dextran solution at a space velocity defined as the permeation rate divided by membrane volume of 6 per hour.

Ann Biomed Eng, 2001 Dec, 29(12), 1100 - 5
Blood cell adhesion on sensor materials studied by light, scanning electron, and atomic-force microscopy; Hildebrand G et al.; Unwanted interactions of biomedical sensors with surrounding tissues, body fluids, and cells are one of the most crucial problems affecting their long-term stability . In vivo processes were simulated in a computer-controlled bioreactor connected to a flow chamber system . Optical sensor materials were inserted into a parallel-plate chamber and monitored by light microscopy in order to get information about the number of adhered cells . Tests with thrombocyte-enriched plasma show that novel phosphorylcholine (PC)-polymer-coated sensors appear to be more bioinert, and thus demonstrate better haemocopatibility in comparison with untreated glass sensors . The influence of different materials on the morphology of adhered cells was investigated by off-line methods such as scanning electron microscopy (SEM) and atomic-force microscopy (AFM) . SEM showed a reduction in the number of adhered thrombocytes and the lack of any fibrin network on the PC-polymer-modified glass surface, in contrast to the pure glass surface . AFM gives topographical information, and the calculated contact areas and cell volumes indicate smaller interactions between cells and sensor material in the case of PC-polymer-coated sensors.

Water Res, 2002 Feb, 36(4), 1018 - 24
Application of a membrane bioreactor for treating explosives process wastewater; Zoh KD et al.; A bench-scale anoxic membrane bioreactor (MBR) system, consisting of a bioreactor coupled to a ceramic cross-flow ultrafiltration module, was evaluated to treat a synthetic wastewater containing alkaline hydrolysis byproducts (hydrolysates) of RDX . The wastewater was formulated the same as hydrolysis wastewater and consisted of acetate, formate and formaldehyde as carbon sources and nitrite and nitrate electron acceptors . The MBR system removed 80-90% of the carbon sources, and approximately 90% of the stoichiometric amount of nitrate, 60% of nitrite . The reactor was also operated over a range of transmembrane pressure, temperature, suspended solids concentration, and organic loading rate to maximize treatment efficiency and permeate flux . Increasing the transmembrane pressure and temperature did not improve flux significantly . Increasing mixed liquor volatile suspended solids (MLVSS) concentration in the bioreactor decreased the permeate flux significantly . The maximum volumetric organic loading rate was 0.72 kg COD/m3/day . The maximum food-to-mass ratio was 0.50 kg N/kg MLVSS/day and 1.82 kg COD/kg MLVSS/day . Membrane permeate was clear and essentially free of bacteria, as indicated by heterotrophic plate count . Permeate flux ranged between 0.15 and 2.0 m3/m2 day and was maintained by routine backwashing every three days . Backwashing with tap water containing chlorine bleach every fourth or fifth backwashing was able to restore membrane flux to its original value.

Environ Toxicol, 2002 Feb, 17(1), 40 - 8
Inhibition of sulfate-reducing bacteria by metal sulfide formation in bioremediation of acid mine drainage; Utgikar VP et al.; Acid mine drainage (AMD) containing high concentrations of sulfate and heavy metal ions can be treated by biological sulfate reduction . It has been reported that the effect of heavy metals on sulfate-reducing bacteria (SRB) can be stimulatory at lower concentrations and toxic/inhibitory at higher concentrations . The quantification of the toxic/inhibitory effect of dissolved heavy metals is critical for the design and operation of an effective AMD bioremediation process . Serum bottle and batch reactor studies on metal toxicity to SRB indicate that insoluble metal sulfides can inhibit the SRB activity as well . The mechanism of inhibition is postulated to be external to the bacterial cell . The experimental data indicate that the metal sulfides formed due to the reaction between the dissolved metal and biogenic sulfide act as barriers preventing the access of the reactants (sulfate, organic matter) to the necessary enzymes . Scanning electron micrographs of the SRB cultures exposed to copper and zinc provide supporting evidence for this hypothesis . The SRB cultures retained their ability to effect sulfate reduction indicating that the metal sulfides were not lethally toxic to the SRB . This phenomenon of metal sulfide inhibition of the SRB has to be taken into account while designing a sulfate-reducing bioreator, and subsequently an efficient biotreatment strategy for AMD . Any metal sulfide formed in the bioreactor needs to be removed immediately from the system to maintain the efficiency of the process of sulfate reduction.

Trends Biotechnol, 2002 Mar, 20(3), 99 - 102
Membrane anchors for vacuolar targeting: application in plant bioreactors; Jiang L et al.; Transgenic plants are attractive expression systems for producing recombinant proteins . Plant cells compartmentalize and store metabolites and proteins in vacuoles, but foreign proteins need to be targeted to the correct compartments if they are to accumulate in a stable fashion . Here we present a general strategy in which unique transmembrane and cytoplasmic tail sequences are used as anchors for delivering recombinant proteins via distinct vesicular transport pathways to specific vacuolar compartments where stable accumulation can occur.

Dev Dyn, 2002 Mar, 223(2), 285 - 91
A critical period for functional vestibular development in zebrafish; Moorman SJ et al.; We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture . A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits . Zebrafish eggs were collected within 3 hours of being laid and fertilized . In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF . In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF . Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days . Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days . These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF . To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF . VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF . These larvae had VOR deficits that persisted for at least 5 days . In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits . In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF . These larvae had normal VORs when they were removed from the bioreactor at 96 hPF . Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system . The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections .

Science, 2002 Feb 8, 295(5557), 1005 - 9
A bioartificial liver--state of the art; Strain AJ et al.; End-stage liver disease is treated by liver transplantation, but donor organ shortages remain a serious problem . This has prompted the design of bioartificial liver devices to "bridge" patients until they either recover or receive a liver transplant . In these devices, patient plasma is circulated extracorporeally through a bioreactor that houses liver cells (hepatocytes) sandwiched between artificial plates or capillaries.

Biotechnol Appl Biochem, 2002 Feb, 35(Pt 1), 19 - 26
High-level expression of human thyroid-stimulating hormone in Chinese hamster ovary cells by co-transfection of dicistronic expression vectors followed by a dual-marker amplification strategy; Peroni CN et al.; The utilization of dicistronic mRNA expression vectors, containing the gene of interest upstream of an amplifiable marker gene, has shown success in rapidly, efficiently and reproducibly obtaining stable cell lines that express high levels of the protein of interest . For this reason, human thyroid-stimulating hormone (hTSH), a heterodimeric glycoprotein composed of non-covalently linked alpha- and beta-subunits, was expressed in Chinese hamster ovary (CHO) cells using a system based on dicistronic expression vectors . These contained the genes of interest and the amplifiable gene markers dihydrofolate reductase (DHFR) and adenosine deaminase (ADA), separated by an internal ribosome entry site isolated from the encephalomyocarditis virus . After the cells (CHO-DHFR-) had been co-transfected with the expression vectors and submitted to gene amplification in culture medium containing stepwise increments of methotrexate, it was possible to isolate clones that presented a secretion level of up to 7.2+/-1.3 microg/10(6) cells per day, the highest ever reported for the expression of this glycoprotein hormone . A second treatment, involving the utilization of deoxycoformycin, directed to amplify the ADA marker gene, provided a clone with an additional 2-3-fold increase in hTSH secretion, reaching a secretion level of 17.8+/-7.6 microg/10(6) cells per day . Cell culture and hTSH production in a hollow-fibre bioreactor were set up in order to carry out a preliminary physico-chemical, immunological and biological characterization of this hormone in comparison with pituitary-extracted hTSH (from the National Institute of Diabetes and Digestive and Kidney Diseases) and the only recombinant hTSH now available (Thyrogen) . The availability of recombinant hTSH is very important in the diagnosis and therapy of thyroid carcinoma, via stimulation of radioiodine uptake.

Rev Argent Microbiol, 2001 Oct-Dec, 33(4), 223 - 8
{Degradation of anthraquinone blue by Trametes trogii}; Levin L et al.; The ability of the white rot fungus Trametes trogii BAFC 463 (high producer of ligninolytic enzymes, especially laccase and manganese peroxidase) to degrade the dye anthraquinone blue, refractory to bacterial attack, was evaluated . Both tropho- and idiophasic T . trogii cultures in synthetic medium (glucose/asparagine) and complex medium (malt extract/glucose) were able to transform up to 88% dye in 4 hours . The activity of laccase, an oxygen-dependent phenoloxidase which was present at high levels in all the conditions assayed, might be related to the ability of the fungus to degrade the colorant . This is supported by the fact that in bioreactor experiences carried out at pH 4.5 the addition of anthraquinone blue caused a decrease in the levels of soluble oxygen . However, although high levels of laccase were produced at pH 7.5, the enzyme was not active, and neither dye transformation nor loss in the levels of soluble oxygen were quantified.

Water Res, 2002 Jan, 36(2), 370 - 93
Modern scientific methods and their potential in wastewater science and technology; Wilderer PA et al.; Application of novel analytical and investigative methods such as fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), microelectrodes and advanced numerical simulation has led to new insights into micro- and macroscopic processes in bioreactors . However, the question is still open whether or not these new findings and the subsequent gain of knowledge are of significant practical relevance and if so, where and how . To find suitable answers it is necessary for engineers to know what can be expected by applying these modern analytical tools . Similarly, scientists could benefit significantly from an intensive dialogue with engineers in order to find out about practical problems and conditions existing in wastewater treatment systems . In this paper, an attempt is made to help bridge the gap between science and engineering in biological wastewater treatment . We provide an overview of recently developed methods in microbiology and in mathematical modeling and numerical simulation . A questionnaire is presented which may help generate a platform from which further technical and scientific developments can be accomplished . Both the paper and the questionnaire are aimed at encouraging scientists and engineers to enter into an intensive, mutually beneficial dialogue.

Water Res, 2002 Feb, 36(3), 638 - 46
Stability of the bacterial communities supported by a seven-stage biological process treating pharmaceutical wastewater as revealed by PCR-DGGE; LaPara TM et al.; The stabilities of the bacterial community structures supported by seven full-scale biological reactors treating pharmaceutical wastewater were investigated by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments . Effluent quality from this treatment process was consistently high with respect to BOD5 (<30 mgl(-1)), soluble COD (<500 mgl(-1)), and total ammonia (< 5 mgl(-1) as N) concentrations . Long-term community structure stability was studied by comparing the similarity of PCR-DGGE fingerprints from samples collected 87 days apart between which the influent wastewater characteristics were relatively stable . The Dice index (Cs) of similarity was moderately high for the first four reactors (Cs = 0.61-0.77) and very high for the last three reactors (Cs = 0.89-0.91) . Short-term community structure stability was studied by comparing PCR-DGGE fingerprints from samples collected 15 days apart between which the influent wastewater characteristics changed significantly, while the effluent quality remained consistently high . The bacterial community composition of each of the seven bioreactors showed a moderate community shift (Cs = 0.70-0.76) . Short-term variability in influent wastewater composition, therefore, affected a greater community shift than did long-term operation treating a wastewater of relatively consistent composition . These results indicate that functionally stable wastewater treatment bioreactors have stable microbial community structures under normal operating conditions but are able to adapt in response to perturbations to sustain high effluent quality.

Hepatology, 2002 Feb, 35(2), 315 - 24
Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats; Yin L et al.; Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected . These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings . When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors . In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel . Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction.

Facial Plast Surg, 2002 Feb, 18(1), 59 - 68
Tissue engineering with chondrocytes; Koch RJ et al.; Tissue engineering of cartilage, using chondrocytes based on the use of synthetic biodegradable polymer cell delivery vehicles (scaffolds), is an alternate treatment modality for replacing missing cartilage . Cartilage tissue engineering has an important role to play in the generation of graft material for head and neck reconstruction . It is an approach to fabricate cartilage constructs in vitro, which could be used in reconstructive surgery . Methods involve (1) harvesting septal cartilage during septoplasty, (2) isolating chondrocytes through enzymatic digestion of the septal cartilage, (3) expanding the cell number in a two-dimensional monolayer culture, using serum-free media, (4) seeding the cells onto a biodegradable polymer scaffold, and (5) cultivating the seeded scaffolds in a rotating bioreactor . In this article we briefly outline the methodology and clinical applications of cartilage grown ex vivo.

Biotechnol Prog, 2002 Jan-Feb, 18(1), 62 - 71
Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion; Huang YM et al.; Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites . This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga . Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied . A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8 . Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter . Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered . Batch cultivation was phosphate-limited in ASP12 artificial seawater medium . However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled . The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times . In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture . Model predictions agreed reasonably with the measured growth curves.

Curr Microbiol, 2002 Mar, 44(3), 173 - 7
Immobilization results in sustained calcium transport in Nostoc calcicola Bréb; Pandey PK et al.; The uptake pattern of Ca2+ by the cyanobacterium Nostoc calcicola Breb in its freely suspended and immobilized form is comprised of two distinct phages; (a) rapid uptake for 1st 10 min followed by (b) slower transport at least up to 60 min . Entrapment of cyanobacterial cells in polyvinyl foam always maintained a higher Ca2+ profile over freely suspended cells . Also, the intracellular Ca2+ concentration was three times more in the former under similar experimental conditions . Whereas, illumination supported maximum Ca2+ transport in all the sets, darkness resulted in drastic reduction (90%) of Ca2+ uptake in freely suspended cells and least (15%) in polyvinyl entrapped cyanobacterial cells . Exogenously added ATP (10 microM) on the other hand, enhanced Ca2+ uptake in dark incubated freely suspended cells; ATP at the same concentration failed to bring out any significant enhancement in cation uptake in immobilized cells facing dark exposure . It was observed that these cells were still able to sustain sufficient ATP preserves to drive active transport of Ca2+ even in the dark . Furthermore, the immobilized cells exhibited remarkable Ca2+ transport rate even at the age of 20 and 50 days at which its free living counterpart took up insignificant Ca2+ . These findings suggest the improved metabolic efficiency of polyvinyl foam entrapped cells over freely suspended cells in terms of Ca2+ accumulation and its possible use as a bioreactor for metal accumulation/removal in repetitive cycles without any measurable loss in cell biomass.

World J Gastroenterol, 1999 Apr, 5(2), 135 - 137
Experimental study of bioartificial liver with cultured human liver cells; Wang YJ et al.; AIM:To establish an extracorporeal bioartificial liver support system (EBLSS) using cultured human liver cells and to study its support effect for fulminant hepatic failure (FHF).METHODS:The liver support experiment of EBLSS consisting of aggregates cultured human liver cells, hollow fiber bioreactor, and circulation unit was carried out in dizhepatic dogs.RESULTS:The viability of isolated hepatocytes and nonparenchymal liver cells reached 96% . These cells were successfully cultured as multicellular spheroids with synthetic technique . The typical morphological appearance was retained up to the end of the artificial liver experiment . Compared with the control dogs treated with EBLSS without liver cells, the survival time of artificial liver support dogs was significantly prolonged . The changes of blood pressure, heart rate and ECG were slow . Both serum ammonia and lactate levels were significantly lowered at the 3rd h and 5th h . In addition, a good viability of human liver cells was noted after 5h experiment.CONCLUSION:EBLSS playing a metabolic role of cultured human hepatocytes,is capable of compensating the function of the liver, and could provide effective artificial liver support and therapy for patients with FHF.

Environ Technol, 2001 Sep, 22(9), 1035 - 42
Application of surface modified polypropylene membranes to an anaerobic membrane bioreactor; Sainbayar A et al.; In order to increase hydrophilicity and thereby to reduce membrane fouling caused by hydrophobic adsorption, the surface of a hydrophobic 0.2 microm polypropylene (PP) membrane was modified by ozone treatment followed by graft polymerization with 2-hydroxy-ethyl methacrylate (HEMA) . The modified PP (MPP) membranes were characterized in terms of contact angle, morphology and degree of grafting (DG) . The contact angle was reduced from 112 degrees for a PP membrane to nearly 0 degrees for MPP membranes by introducing functional groups such as hydroxyl (-OH) and carbonyl groups (C=O) on the membrane surface . As the DG increased, the O/C ratio and membrane resistance of the MPP membrane increased . Using the MPP membrane in the crossflow operation of an anaerobic membrane bioreactor (MBR), the membrane permeability was enhanced although it was largely dependent on the DG of MPP.

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