J Biotechnol, 2001 Dec 14, 92(1), 47 - 59 Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression; Carvalhal AV et al.; Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth . With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability . To apply this concept in industry, a reduction of the time span of estradiol exposure is required . Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48-72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day) . Cell growth inhibition was successfully achieved for three consecutive cycles . Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used . These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM . Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied . This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent . This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes. Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 796 - 802 Microbial degradation of phenanthrene and pyrene in a two-liquid phase-partitioning bioreactor; Guieysse B et al.; A study was conducted to determine the potential of two-liquid phase-bioreactors for the treatment of (polycyclic aromatic hydrocarbons) PAHs . Phenanthrene and pyrene were supplied two times at a concentration of 100 mg/l of reactor broth, either as crystals or dissolved in silicone oil . Complete phenanthrene biodegradation was achieved within 3 days after each addition to the biphasic-inoculated reactor . Its concentration in the monophasic reactors dropped by 93% within 4 days, but remained incomplete for the duration of the experiment . Pyrene removal occurred to a limited extent only in the presence of phenanthrene . Significant pollutant losses were recorded in the monophasic reactors, most likely caused by volatilization . Pollutant degradation was improved upon repeated phenanthrene amendment to the biphasic system . Biphasic reactors allow the fast and complete degradation of PAHs and prevent their hazardous disappearance . The use of biphasic reactors for the degradation of poorly soluble pollutants should become more beneficial when the substrate-interface uptake mechanism is operating . Thus, biphasic reactors should be integrated into the microbial enrichment procedure. Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 780 - 7 Biomonitoring of continuous microbial community adaptation towards more efficient phenol-degradation in a fed-batch bioreactor; Guieysse B et al.; The anaerobic degradation of phenol was studied in a fed-batch culture . Nitrate was added as electron acceptor and phenol was provided three times, to a final concentration of 200 mg/l . Randomly amplified polymorphic DNA (RAPD) and terminal fraction fragment length polymorphism (T-RFLP) were used and compared in order to monitor the microbial succession in the reactor . Phenol degradation started after an initial lag phase of 14 days and was then completed within a few days . In addition, the duration of the lag phase was shortened and the degradation rate was increased after each phenol amendment . Nitrate reduction correlated with microbial growth and phenol depletion, confirming that the degradation was carried out anaerobically . Results from the DNA analysis showed that the structure of the microbial community changed after each phenol amendment . This study confirms the potential for anaerobic degradation of environmental pollutants and also confirms that microbial acclimation towards faster degradation rates occurred upon repeated substrate amendments . Furthermore, both of the DNA-based techniques described the phenol degradation-linked community shifts with similar general results . RAPD is a faster, simpler technique that gives a higher resolution and consequently reflects the shifts in the microbial community structure better, whereas T-RFLP is more suitable for phylogenetic studies. Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 623 - 33 Mass production of entomopathogenic nematodes for plant protection; Ehlers RU; Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are commercially used to control pest insects . They are symbiotically associated with bacteria of the genera Photorhabdus and Xenorhabdus, respectively, which are the major food source for the nematodes . The biology of the nematode-bacterium complex is described, a historical review of the development of in vitro cultivation techniques is given and the current use in agriculture is summarised . Cultures of the complex are pre-incubated with the symbiotic bacteria before the nematodes are inoculated . Whereas the inoculum preparation and preservation of bacterial stocks follow standard rules, nematodes need special treatment . Media development is mainly directed towards cost reduction, as the bacteria are able to metabolise a variety of protein sources to provide optimal conditions for nematode reproduction . The process technology is described, discussing the influence of bioreactor design and process parameters required to obtain high nematode yields . As two organisms are grown in one vessel and one of them is a multicellular organism, the population dynamics and symbiotic interactions need to be understood in order to improve process management . Major problems can originate from the delayed or slow development of the nematode inoculum and from phase variants of the symbiotic bacteria that have negative effects on nematode development and reproduction . Recent scientific progress has helped to understand the biological and technical parameters that influence the process, thus enabling transfer to an industrial scale . As a consequence, costs for nematode-based products could be significantly reduced. Ann Biomed Eng, 2001 Sep, 29(9), 752 - 63 Biosynthetic activity in heart valve leaflets in response to in vitro flow environments; Weston MW et al.; The development of bioreactors for tissue engineered heart valves would be aided by a thorough understanding of how mechanical forces impact cells within valve leaflets . The hypothesis of the present study is that flow may influence the biosynthetic activity of aortic valve leaflet cells . Porcine leaflets were exposed to one of several conditions for 48 h, including steady or pulsatile flow in a tubular flow system at 10 or 20 l/min, and steady shear stress in a parallel plate flow system at 1, 6, or 22 dyne/cm2 . Protein, glycosaminoglycan, and DNA synthesis increased during static incubation but remained at basal levels after exposure to flow . The modulation of synthetic activity was attributed to the presence of a shear stress on the leaflet surface, which may be transmitted to cells within the leaflet matrix through tensile forces . The alpha-smooth muscle (alpha-SM) actin distribution observed in fresh leaflets was proportionately decreased after exposure to antibiotics and not recovered by either static incubation or exposure to flow . These results indicate that exposure to flow maintains leaflet synthetic activity near normal levels, but that the inclusion of another force, such as bending or backpressure, may be necessary to preserve alpha-SM actin immunoreactive cells. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001 Sep, 36(8), 1549 - 58 Feasibility of on-site bioremediation of loam soil contaminated by diesel oil; Rubin H et al.; This study originated from an accidental event of diesel oil contamination in a loam soil area of 7,000 m2 . Approximately a volume of 1,300 m3 of diesel oil was released into the environment . Reclamation of the contaminated soil by on-site bioremediation was selected as the most appropriate treatment method . A major concern was associated with the nature of the local loam soil . Loam has a very low hydraulic conductivity and very quickly becomes impermeable after its contact with water . The bioremediation approach incorporated excavation of the contaminated soil, mixing it with an agent, which increased its permeability . Following this preliminary treatment came the construction of bioreactors as a suitable environment of nutrients, moisture, dissolved oxygen, and enriched culture of microorganisms, which enabled breakdown of the diesel oil . This case study indicated that the target of 99% of diesel oil clean up could be achieved by using the technology of on-site bioremediation . The selected treatment method was found to be technologically and economically feasible . However, some improvement in the application of the basic treatment approach might increase the bioremediation efficiency. Magn Reson Med, 2001 Oct, 46(4), 819 - 26 EPR oxygen mapping (EPROM) of engineered cartilage grown in a hollow-fiber bioreactor; Ellis SJ et al.; A novel electron paramagnetic resonance (EPR)-based oxygen mapping procedure (EPROM) is applied to cartilage grown in a single-, hollow-fiber bioreactor (HFBR) system . Chondrocytes harvested from the sterna of 17-day-old chick embryos were inoculated into an HFBR and produced hyaline cartilage over a period of 4 weeks . Tissue oxygen maps were generated according to the EPROM technique (Velan et al., Magn Reson Med 2000;43:804-809) by making use of the line-broadening effects of oxygen on the signal generated from nitroxide spin probes . In addition, the effect on oxygen consumption of the addition of cyanide to the tissue was investigated . Cyanide is a potent inhibitor of oxidative phosphorylation, and accordingly, given the constant provision of oxygen to the tissue, it would be expected to increase oxygen levels within the HFBR . The EPROM measurements showed a significant increase in oxygen concentration in the cartilage after the addition of cyanide . In contrast to other methods for studying oxygen in cartilage, EPROM can provide direct, noninvasive visualization of local concentrations in three dimensions . Biotechnol Bioeng, 2001 Nov 5, 75(3), 379 - 85 Surface transformation of bioactive glass in bioreactors simulating microgravity conditions . Part II: numerical simulations; Gao H et al.; The effects of simulated microgravity on the surface modification of bioactive glass (BG) in solution were studied using a numerical method . Models were developed for estimating the mass transfers of different chemical species from the surface of bioactive glass particles (microcarriers) suspended in the rotating liquid medium of a NASA-designed high aspect ratio vessel (HARV) bioreactor and on the bottom surface of a static vial . The concentration profiles resulting from chemical reactions and ionic transports were ascertained . Numerical results for the transport under simulated microgravity in the HARV and at normal gravity in the static vial were compared . These results were also compared with those of experiments to verify the enhancement of the reaction kinetics under simulated microgravity conditions . The experimental and numerical studies confirm that simulated microgravity conditions lead to the quick achievement of bioactive glass surface modification . Biotechnol Bioeng, 2001 Nov 5, 75(3), 369 - 78 Surface transformation of bioactive glass in bioreactors simulating microgravity conditions . Part I: experimental study; Radin S et al.; Surface modified bioactive glass with surface properties akin to those of the bone mineral phase is an attractive candidate for use as a microcarrier material for 3-D growth of bone-like tissue in rotating wall vessel bioreactors (RWVs) . The critical surface properties of this material are the result of reaction in solution . Because an RWV environment is completely different from conditions previously employed for bioactive glass testing, a detailed study of the surface reactions is warranted . Under properly chosen conditions, RWVs can also provide a simulated microgravity environment for the bioactive glass (BG) particles . In this sense, this study is also a report on the behavior of a bioactive material under microgravity conditions simulated on earth . A high aspect ratio vessel (HARV) and carefully selected experimental conditions enabled the simulation of microgravity in our laboratory . A complimentary numerical study was simultaneously conducted to ascertain the appropriateness of the experimental parameters (particle size, particle density, medium density, medium viscosity, and rotational speed) that ensure simulated microgravity conditions for the glass particles in the HARV . Physiological solutions (pH 7.4) with and without electrolytes, and also with serum proteins, were used to study the change in surface character resulting from simulated microgravity . Control tests at normal gravity, both static and dynamic, were also conducted . Solution and surface analyses revealed major effects of simulated microgravity . The rates of leaching of constituent ions (Si-, Ca-, and P-ions) were greatly increased in all solutions tested . The enhanced dissolution was followed by the enhanced formation of bone-like minerals at the BG surface . This enhancement is expected to affect adsorption of serum proteins and attachment molecules, which, in turn, may favorably affect bone cell adhesion and function . The findings of the study are important for the use of bioactive materials as microcarriers to generate and analyze 3-D bone-like tissue structures in bioreactors under microgravity conditions or otherwise . Copyright John Wiley & Sons, Inc. Biotechnol Bioeng, 2001 Nov 5, 75(3), 355 - 61 Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate; Eriksen NT et al.; This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli . Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH . When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically . The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension . During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension . The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD . Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period . Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions . The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased . No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced . These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction . Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth . Biotechnol Prog, 2001 Sep-Oct, 17(5), 951 - 7 Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry; Lu Chau T et al.; The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins . The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here . This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L . The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction . Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry . This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample . A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique . Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population . Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%) . Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain. Biotechnol Prog, 2001 Sep-Oct, 17(5), 881 - 6 Downstream processing of enzymatically produced geranyl glucoside; de Roode BM et al.; Geraniol plays an important role in the fragrance and flavor industry . The corresponding glucoside has interesting properties as a "slow release" aroma compound . Therefore, the enzymatic production and downstream processing of geranyl glucoside were investigated . Geranyl glucoside was produced in a spray column reactor with an initial production rate of 0.58 mg x U(-1) x h(-1) . A pretreated hydrophobic microfiltration membrane was used to prevent migration of the aqueous, enzyme-containing phase to the downstream process . No retention of the glucoside, which accumulated in the geraniol phase, was found . On the basis of examples from the literature, four downstream processes were tested on their viability for this system . Extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol . In the first case, the glucoside selectivity for the geraniol phase was found to be high, which made extraction with water unsuccessful . In the second case it was possible to obtain a stable foam, but significant enrichment of the foam with glucoside did not occur . Adsorption on alumina and distillation under reduced pressure were applied successfully and tested in-line with the bioreactor . A maximum glucoside adsorption of 7.86 mg x g(-1) was achieved on alumina . After desorption and evaporation of the extractant the pure glucoside was obtained quantitatively . A pure product could not be obtained after distillation because a small amount of glucose was present in the permeate as well, which accumulated in the bottom fraction . It was shown that with this reactor system a production of 1 kg of geranyl glucoside in 2 days is possible using an initial amount of 50,000 units of enzyme. Biotechnol Prog, 2001 Sep-Oct, 17(5), 838 - 46 Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors; Hu WW et al.; A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor . High-density bioreactor batch cultivation was carried out using a modified MS medium . The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each . The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each . Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d) . The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide) . This work successfully demonstrated the high-density bioreactor cultivation of P . notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors . The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species. Biotechnol Prog, 2001 Sep-Oct, 17(5), 828 - 31 New miniaturized hollow-fiber bioreactor for in vivo like cell culture, cell expansion, and production of cell-derived products; Gloeckner H et al.; We have developed a miniaturized hollow-fiber bioreactor system for mammalian cell culture with a volume of 1 mL . Cell and medium compartments of the bioreactor are separated by a semipermeable membrane, and oxygenation of the cell compartment is accomplished using an oxygenation membrane . As a result of the geometry of the transparent housing, cells can be observed by microscopy during culture . The leukemic cell lines CCRF-CEM, HL-60, and REH were cultivated up to densities of 3.5 x 10(7)/mL without medium change or manipulation of the cells . As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate . As a consequence, consumption of glucose and generation of lactate varied with flow rate . Depending on the molecular size cutoff of the membranes used, added growth factors such as GM-CSF, as well as factors secreted from the cells, are retained in the cell compartment for up to 1 week . This new miniaturized hollow-fiber bioreactor offers advantages in tissue engineering by continuous nutrient supply for cells in high density, retention of added or autocrine produced factors, and undisturbed long-term culture in a closed system. Biotechnol Prog, 2001 Sep-Oct, 17(5), 822 - 7 Production of a sialylated N-linked glycoprotein in insect cells; Joshi L et al.; Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri- and tetra-antennary complex N-linked oligosaccharides containing terminal sialic acid residues . The oligosaccharide structures were similar to those produced in human placental cells . Insect cells cultured in T-flasks only performed incomplete oligosaccharide processing . The mechanism of HARV-mediated changes in the eukaryotic N-linked glycosylation pathway was investigated and could be mimicked under T-flask growth conditions with the addition of N-acetylmannosamine to the culture medium . The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight. Biotechnol Prog, 2001 Sep-Oct, 17(5), 809 - 21 Production and molecular characterization of clinical phase i anti-melanoma mouse IgG3 monoclonal antibody R24; Kemminer SE et al.; R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin . The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma . The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody . Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode . The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography . Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains . Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis . Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry . Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation . The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated) . The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated) . The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06 . Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore) . From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy. Biomol Eng, 2001 Oct 31, 18(4), 143 - 77 Enzymes inside lipid vesicles: preparation, reactivity and applications; Walde P et al.; There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space . This has been shown by many investigations carried out with a variety of enzymes . A review of these studies is given and some of the main results are summarized . With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) . Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine) . The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers . If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-rehydration method', followed by the 'extrusion technique' has shown to be superior over other procedures . In addition to many investigations in the field of cheese production--there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g . in the enzyme-replacement therapy or for immunoassays)--including a few in vivo studies . In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system . For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous . Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase . Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products . Using physical (e.g . microwave irradiation) or chemical methods (e.g . addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent . A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there . There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates . Using micrometer-sized vesicles (so-called 'giant vesicles') as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules. ASAIO J, 2001 Sep-Oct, 47(5), 471 - 5 Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients; Mazariegos GV et al.; A Phase I clinical safety evaluation of the Excorp Medical, Inc, Bioartificial Liver Support System (BLSS) is in progress . Inclusion criteria are patients with acute liver failure of any etiology, presenting with encephalopathy deteriorating beyond Parson's Grade 2 . The BLSS consists of a blood pump, heat exchanger to control blood temperature, oxygenator to control oxygenation and pH, 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 . A single support period evaluation consists of 12 hour extracorporeal perfusion with the BLSS sandwiched between 12 hours of pre (baseline) and 12 hours of post support monitoring . Blood chemistries and hematologies are obtained every 6 hours during monitoring periods and every 4 hours during perfusion . Physiologic parameters are monitored continuously . The patient may receive a second treatment at the discretion of the clinical physician . Preliminary evaluation of safety considerations after enrollment of the first four patients (F, 41, acetaminophen induced, two support periods; M, 50, Wilson's disease, one support period; F, 53, acute alcoholic hepatitis, two support periods; F, 24, chemotherapy induced, one support period) is presented . All patients tolerated the extracorporeal perfusion well . All patients presented with hypoglycemia at the start of perfusion, treatable by IV dextrose . Transient hypotension at the start of perfusion responded to an IV fluid bolus . Only the second patient required heparin anticoagulation . No serious or unexpected adverse events were noted . Moderate biochemical response to support was noted in all patients . Completion of the Phase I safety evaluation is required to fully characterize the safety of the BLSS. Theriogenology, 2001 Sep 1, 56(4), 545 - 56 Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats; Huang SZ et al.; The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology . However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low . This may be attributed in part to the low integration rate of foreign DNA . Therefore, a large number of recipients are required to produce transgenic animals . We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats . Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats . In the end, four live-born kids were produced . As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection . The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat . We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale. Huan Jing Ke Xue, 2001 Jul, 22(4), 91 - 4 {Treatment of dying wastewater from a woolen mill with a pilot-scale anaerobic/oxic membrane bioreactor (A/O MBR)}; Zheng X et al.; A pilot-scale (10 t/d) anaerobic/oxic membrane bioreactor (A/O MBR) was used for treatment of dying wastewater from a woolen mill . The results showed that when COD, BOD5 and color in the influent was 179-358 mg/L, 44.8-206 mg/L and 50-240 dilution times (DT), the average COD, BOD5 and color of A/O MBR effluent was 20.2 mg/L, 1.6 mg/L, 25 DT respectively . The removal of COD, BOD5, color, turbidity was 92.1%, 98.4%, 60.7% and 98.9% respectively . Each quota of the treated water met the gray water standards (CJ25.1-89) . The A/O MBR process has many advantages, such as stable performance, simple operation, easy management etc . The result of this work could be reference for the designing of industrial scale A/O MBR process for treatment of the woolen mill wastewater. J Hazard Mater, 2001 Oct 12, 87(1-3), 259 - 71 Impact of various leachate recirculation regimes on municipal solid waste degradation; San I et al.; Landfilled municipal solid waste can be treated by introducing leachate into the waste matrix . Increasing attention is being given to landfill leachate recirculation as a means for in situ leachate treatment and landfill stabilization . Landfills with leachate recirculation may be operated as municipal solid waste bioreactor treatment system rather than as a conventional waste dumping sites . In order to study the impact of various leachate recirculation regimes on municipal solid waste degradation, two landfill-simulating reactors, one with leachate recycle and one without, were constructed and placed at a constant room temperature (34 degrees C) . Both reactors were filled with a municipal solid waste mixture representing the typical solid waste composition determined for the city of Istanbul . For the purpose of this experiment, leachate recirculation volume and frequency were changed periodically . This research showed that increased frequency of leachate recirculation accelerates the stabilization rate of waste matrix . About 2l of recirculated leachate and four times per week recirculation strategy were found to provide the highest degree of waste stabilization . Additionally, this research confirmed that leachate recirculation is a very feasible way for in situ leachate treatment. J Microbiol Methods, 2001 Oct, 47(1), 51 - 7 An improved gas distribution system for anaerobic screening of multiple microbial cultures; Valadi H et al.; A cultivation set-up for multiple cultures has been designed that can be used for anaerobic screening for quantitative changes in growth rate or other analyses, e.g . protein composition of different strains . The developed gas distribution system provides a reproducible level of anaerobicity in 30 cultivation flasks and resembles the open system of a high-performance bioreactor in that it ensures cultivation at atmospheric pressure and avoids supersaturation of carbon dioxide . The system is cheap and user-friendly and allows rapid screenings of many strains simultaneously. Water Res, 2001 Oct, 35(15), 3605 - 10 Biological conversion of hydrogen sulphide to elemental sulphur in a fixed-film continuous flow photo-reactor; Henshaw PF et al.; The green sulphur bacterium Chlorobium thiosulfutophilum was used to remove hydrogen sulphide from synthetic industrial wastewater and convert it to elemental sulphur in a fixed-film continuous-flow photosynthetic bioreactor . Twenty 150 mm x 3 mm ID Tygon tubes formed the active part of the reactor resulting in a total volume of 21.2 mL . Seven steady states were achieved under different experimental conditions using this tubular photo-reactor . Sulphide loading rates ranged from 111 to 328 mg/hL under influent flowrates of 9.0-42.4 mL/h and hydraulic retention times of 0.50-2.35 h . The irradiance at the reactor surface averaged 25.4 W/m2 . The sulphide removal rates were found to be 82-100% and elemental sulphur recovery rates were found to be 75-95% . The maximum sustainable sulphide loading rate was found to be 286 mg/h L, which is 2.5 times higher than the previous reported highest value . Sulphide loading rate was found to be the function of radiant flux per unit reactor volume and the bacteriochlorophyll concentration as expressed by the van Niel curve. Curr Opin Biotechnol, 2001 Aug, 12(4), 419 - 25 Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis; Duetz WA et al.; The unique catalytic properties of oxygenases (the regio-specific and/or enantio-specific hydroxylation of non-activated carbons) are of undisputed biosynthetic value . Factors that govern the economics of their industrial use include a low k(cat), a frequently decreased k(cat) in recombinant strains, limiting oxygen transfer rates in bioreactors, product inhibition, and the demanding discovery (screening) process. Curr Opin Biotechnol, 2001 Aug, 12(4), 411 - 8 Producing proteins in transgenic plants and animals; Larrick JW et al.; The requirement for large quantities of therapeutic proteins has fueled interest in the production of recombinant proteins in plants and animals . The first commercial products to be made in this way have experienced much success, and it is predicted that in the future a plethora of protein products will be made using these 'natural' bioreactors. Cell Transplant, 2001, 10(4-5), 429 - 33 In vivo estimation of bioartificial liver with recombinant HepG2 cells using pigs with ischemic liver failure; Enosawa S et al.; Biological efficacy of a recombinant human hepatic cell line, glutamine synthetase transfected HepG2 (GS-HepG2), was examined with large-scale culture in a circulatory flow bioreactor and in pigs with ischemic liver failure . GS-HepG2 cells were cultured in a circulatory flow bioreactor from 5 x 10(7) to 4 x 10(9) cells for 109 days . The cells showed ammonia removal activity even under substrate (glutamic acid)-free medium, suggesting that the GS catalyzed the activity using intracellular glutamic acid that had been pooled during conventional culture . When GS-HepG2 bioartificial liver (BAL) was applied to pigs with ischemic liver failure, survival time was prolonged to 18.8 +/- 6.1 h (mean +/- SD, n = 4) from 13.8 +/- 5.4 h (n = 6) and 10.7 +/- 4.1 h (n = 6) (groups treated with cell-free BAL and treated with plasma exchange and continuous hemodiafiltration, respectively) . Laboratory data indicated the tendency for improvement in increase of blood ammonia level and decline of blood coagulation indices in the GS-HepG2 BAL-treated group . The advantages and potential for the cell line as a bioreactor in BAL is also discussed, comparing to those of isolated porcine hepatocytes. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 560 - 5 Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised on sand for the purpose of treating acid mine-drainage; Wood TA et al.; Thiobacillus ferrooxidans was immobilised on sand (size 0.85 mm to 1.18 mm) for use in a repeated batch and continuously operated packed-bed bioreactor which has not been previously reported in the literature . Repeated batch operation resulted in the complete oxidation of ferrous to ferric iron . The bacteria were active immediately after 3-4 weeks in a non-aqueous medium; i.e . the sand was allowed to dry out, demonstrating the stability of the system . A lag phase of 28 days was recorded when the sand was stored dried in a sealed container for 16 weeks compared with a lag phase of 13 days for a sample frozen for 18 weeks . After a period of 10 days, continuous operation of the reactor at a dilution rate of 0.64 h(-1) resulted in 95-99% oxidation of ferrous iron or 0.31-0.33 kg m(-3) h(-1) . With the use of a scanning electron microscope, images were recorded of Thiobacillus ferrooxidans on sand. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 384 - 7 Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not; Gao Q et al.; Stress, including that caused by ethanol, has been shown to induce or promote secondary metabolism in a number of microbial systems . Rotating-wall bioreactors provide a low stress and simulated microgravity environment which, however, supports only poor production of microcin B17 by Escherichia coli ZK650, as compared to production in agitated flasks . We wondered whether the poor production is due to the low level of stress and whether increasing stress in the bioreactors would raise the amount of microcin B17 formed . We found that applying shear stress by addition of a single Teflon bead to a rotating wall bioreactor improved microcin B17 production . By contrast, addition of various concentrations of ethanol to such bioreactors (or to shaken flasks) failed to increase microcin B17 production . Ethanol stress merely decreased production and, at higher concentrations, inhibited growth . Interestingly, cells growing in the bioreactor were much more resistant to the growth-inhibitory and production-inhibitory effects of ethanol than cells growing in shaken flasks. Semin Thromb Hemost, 2001 Aug, 27(4), 405 - 16 Recombinant antithrombin: production and role in cardiovascular disorder; Levy JH et al.; Plasma-derived antithrombin (AT) concentrates have been used for the management of hereditary and acquired deficiencies since the early 1980s . Recombinant versions of other blood factors and their derivatives are increasingly becoming available, providing a safe and abundant supply of these important therapeutics . However, the complexity of the AT molecule and the large doses often required for supplementation treatments preclude the use of traditional cell culture bioreactors for recombinant production . The development of a very efficient expression system has been necessary for the cost-efficient recombinant production of AT . Transgenic production, with its ability to yield high levels of heterologous protein and its scale-up flexibility, is an attractive alternative to plasma fractionation . Purification of recombinant AT from the milk of transgenic dairy goats has been developed to provide a homogeneous, well-defined, and abundant supply of this factor . This article describes the production of recombinant AT and aspects of clinical applications of this molecule to cardiovascular disorders. Adv Space Res, 2000, 26(2), 253 - 62 The C.E.B.A.S.-Minimodule: behaviour of an artificial aquatic ecological system during spaceflight; Bluem V et al.; The C.E.B.A.S.-Minimodule, a closed aquatic ecosystem integrated into a middeck locker and consisting of a Zoological (animal tanks), a Botanical (plant bioreactor), a Microbial (bacteria filter) and an Electronic Component (data acquisition/control system) was flown on the STS-89 spaceshuttle mission in January 1998 for 9 days . Preflight the plant bioreactor was loaded with 53 g of Ceratophyllum demersum (coontail) and the animal tanks with 4 adult pregnant females of the fish, Xiphophorus helleri (sword-tails), 200 juveniles of the same species less than 1 week of age, 38 large and 30 juvenile Biomphalaria glabrata water snails . The filter compartment was filled with 200 g of lava grain inoculated with laboratory strains of ammonia-oxidizing bacteria . A ground reference was undertaken with the same biological setup with a delay of 4 d . After an adaptation period of 5 d the system was closed and integrated into the spaceshuttle one day before launch . Video recordings of the animals were automatically taken for 10 minutes in 2-hour periods; the tapes were changed daily by the astronauts . The chemical and physical data for the aquatic system were within the expected range and were closely comparable in comparison to the ground reference . After 9 d under space conditions, the plant biomass increased to 117 g . The plants were all found in very good condition . All 4 adult female fish were retrieved in a good physiological condition . The juvenile fishes had a survival rate of about 33% . Almost 97% of the snails had survived and produced more than 250 neonates and 40 spawning packs . All samples were distributed according to a defined schedule and satisfied all scientific needs of the involved 12 principal investigators . This was the first successful spaceflight of an artificial aquatic ecosystem containing vertebrates, invertebrates, higher plants and microorganisms self-sustained by its inhabitants only . C.E.B.A.S . in a modified form and biological setup is a promising candidate for the early space station utilization as a first midterm experiment. Adv Space Res, 1999, 24(6), 829 - 36 Cultures of human liver cells in simulated microgravity environment; Yoffe B et al.; We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC) . To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels . Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed . Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions . Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix . Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression . In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions. Adv Space Res, 1998, 22(10), 1453 - 64 Progress in ultrasonic bioreactors for CELSS applications; Schlager KJ; An important issue in Controlled Ecological Life Support Systems (CELSS) is the recycling of inedible crop residues to recover inorganic plant nutrients such as nitrates, phosphates, potassium and other macro- and micro-nutrients . In a closed system in space, such regeneration is vital to the long term viability of plant growth necessary for the food production and waste handling process . Chemical approaches to recycling such as incineration and wet oxidation are not compatible with low energy and environmentally friendly regeneration of such nutrients . Biological regeneration is more acceptable environmentally, but it is a very slow process and does not typically result in complete recovery of inorganic and organic nutrients . A new approach to biological regeneration is described here involving the combined use of special enzymatic catalysts and ultrasonic energy in a bioreactor system . This new system has the potential for rapid, efficient, environmentally friendly and complete conversion of crop wastes to inorganic plant nutrients and food recovery from cellulose materials . A series of experimental tests were carried out with a soybean crop residue meal substrate . Biochemical conversion rates were significantly expedited with the addition of enzymes and further enhanced through ultrasonic stimulation of these enzymes . The difference in conversion rates was particularly increased after the initial period of soluble organics conversion . The remaining cellulose substrate is much more difficult to biodegrade, and the ultrasonically-enhanced reaction was able to demonstrate a much higher rate of substrate conversion. Adv Space Res, 1997, 20(10), 2023 - 8 Effects of bioreactor retention time on aerobic microbial decomposition of CELSS crop residues; Strayer RF et al.; The focus of resource recovery research at the KSC-CELSS Breadboard Project has been the evaluation of microbiologically mediated biodegradation of crop residues by manipulation of bioreactor process and environmental variables . We will present results from over 3 years of studies that used laboratory- and breadboard-scale (8 and 120 L working volumes, respectively) aerobic, fed-batch, continuous stirred tank reactors (CSTR) for recovery of carbon and minerals from breadboard grown wheat and white potato residues . The paper will focus on the effects of a key process variable--bioreactor retention time--on response variables indicative of bioreactor performance . The goal is to determine the shortest retention time that is feasible for processing CELSS crop residues, thereby reducing bioreactor volume and weight requirements . Pushing the lower limits of bioreactor retention times will provide useful data for engineers who need to compare biological and physicochemical components . Bioreactor retention times were manipulated to range between 0.25 and 48 days . Results indicate that increases in retention time lead to a 4-fold increase in crop residue biodegradation, as measured by both dry weight losses and CO2 production . A similar overall trend was also observed for crop residue fiber (cellulose and hemicellulose), with a noticeable jump in cellulose degradation between the 5.3 day and 10.7 day retention times . Water-soluble organic compounds (measured as soluble TOC) were appreciably reduced by more than 4-fold at all retention times tested . Results from a study of even shorter retention times (down to 0.25 days), in progress, will also be presented. Adv Space Res, 1997, 20(10), 1815 - 20 Use of biologically reclaimed minerals for continuous hydroponic potato production in a CELSS; Mackowiak CL et al.; Plant-derived nutrients were successfully recycled in a Controlled Ecological Life Support System (CELSS) using biological methods . The majority of the essential nutrients were recovered by microbiologically treating the plant biomass in an aerobic bioreactor . Liquid effluent containing the nutrients was then returned to the biomass production component via a recirculating hydroponic system . Potato (Solanum tuberosum L.) cv . Norland plants were grown on those nutrients in either a batch production mode (same age plants on a nutrient solution) or a staggered production mode (4 different ages of plants on a nutrient solution) . The study continued over a period of 418 days, within NASA Breadboard Project's Biomass Production Chamber at the Kennedy Space Center . During this period, four consecutive batch cycles (104-day harvests) and 13 consecutive staggered cycles (26-day harvests) were completed using reclaimed minerals and compared to plants grown with standard nutrient solutions . All nutrient solutions were continually recirculated during the entire 418 day study . In general, tuber yields with reclaimed minerals were within 10% of control solutions . Contaminants, such as sodium and recalcitrant organics tended to increase over time in solutions containing reclaimed minerals, however tuber composition was comparable to tubers grown in the control solutions. Adv Space Res, 1999, 24(3), 319 - 28 Development of autonomous control in a closed microbial bioreactor; Smernoff DT et al.; Space-based life support systems which include ecological components will rely on sophisticated hardware and software to monitor and control key system parameters . Autonomous closed artificial ecosystems are useful for research in numerous fields . We are developing a bioreactor designed to study both microbe-environment interactions and autonomous control systems . Currently we are investigating N-cycling and N-mass balance in closed microbial systems . The design features of the system involve real-time monitoring of physical parameters (e.g . temperature, light), growth solution composition (e.g . pH, NOx, CO2), cell density and the status of important hardware components . Control of key system parameters is achieved by incorporation of artificial intelligence software tools that permit autonomous decision-making by the instrument . These developments provide a valuable research tool for terrestrial microbial ecology, as well as a testbed for implementation of artificial intelligence concepts . Autonomous instrumentation will be necessary for robust operation of space-based life support systems, and for use on robotic spacecraft . Sample data acquired from the system, important features of software components, and potential applications for terrestrial and space research will be presented. Chimia (Aarau), 1999 Mar, 53(3), 75 - 80 Microtechnology in space bioreactors; Walther I et al.; Space biology is a young and rapidly developing discipline comprising basic research and biotechnology . In the next decades it will play a prominent role in the International Space Station (ISS) . Therefore, there is an increasing demand for sophisticated instrumentation to satisfy the requirements of the future projects in space biology . Bioreactors will be needed to supply fresh living material (cells and tissues) either to study still obscure basic biological mechanisms or to develop profitable bioprocesses which will take advantage of the peculiar microgravity conditions . Since more than twenty years, the Space Biology Group of the ETHZ is carrying out research projects in space (Space Shuttle/Spacelab, MIR Station, satellites, and sounding rockets) that involve also the development of space-qualified instrumentation . In the last ten years we have developed, in collaboration with Mecanex SA, Nyon, and the Institute of Microtechnology of the University of Neuchatel, a space bioreactor for the continuous culture of yeast cells under controlled conditions . Sensors, pH control, nutrients pump and fluid flowmeter are based on state-of-the-art silicon technology . After two successful space flights, a further improved version is presently prepared for a flight in the year 2000. Gravit Space Biol Bull, 1999 May, 12(2), 57 - 66 Microgravity cultivation of cells and tissues; Freed LE et al.; In vitro studies of cells and tissues in microgravity, either simulated by cultivation conditions on earth or actual, during spaceflight, are expected to help identify mechanisms underlying gravity sensing and transduction in biological organisms . In this paper, we review rotating bioreactor studies of engineered skeletal and cardiovascular tissues carried out in unit gravity, a four month long cartilage tissue engineering study carried out aboard the Mir Space Station, and the ongoing laboratory development and testing of a system for cell and tissue cultivation aboard the International Space Station. Acta Astronaut, 1998 Jan-Apr, 42(1-8), 455 - 63 Taxane recovery from cells of Taxus in micro- and hypergravity; Durzan DJ et al.; Cell suspension cultures of Taxus cuspidata produce taxanes that are released from the outer surface of cells into the culture medium as free and bound alkaloids . Paclitaxel (Taxol (TM)), is an anti-cancer drug in short supply . It has a taxane ring derived from baccatin III and a C-13 phenylisoserine side-chain . This drug is produced over a wide range of gravitational forces . Monoclonal and polyclonal antibodies to paclitaxel, baccatin III, and the C-13 phenylisoserine side chain were combined in multiple-labeling studies to localize taxanes and paclitaxel on cell surfaces or on particles released into the culture medium . Bioreactor vessel design altered the composition of taxanes recovered from cells in simulated microgravity . At 10(-2) and 2x10(-4)g, taxane recovery was reduced but biomass growth and percent paclitaxel was significantly increased . At 1 to 24g, growth was reduced with a significant recovery of total taxanes with low percent paclitaxel . Bound paclitaxel was also localized in endonuclease-rich fragmenting nuclei of individual apoptotic cells . A model is presented comprising TCH (touch) genes encoding enzymes that modify taxane-bearing xylan residues in cell walls, the calcium-sensing of gravitational forces by the cytoplasm, and the predisposition of nuclei to apoptosis . This integrates the adaptive physiological and biochemical responses of drug-producing genomes with gravitational forces. Acta Hortic, 1996 Dec, 440, 19 - 24 Recycling crop residues for use in recirculating hydroponic crop production; Mackowiak CL et al.; As part of bioregenerative life support feasibility testing by NASA, crop residues are being used to resupply elemental nutrients to recirculating hydroponic crop production systems . Methods for recovering nutrients from crop residues have evolved from water soaking (leaching) to rapid aerobic bioreactor processing . Leaching residues recovered the majority of elements but it also recovered significant amounts of soluble organics . The high organic content of leachates was detrimental to plant growth . Aerobic bioreactor processing reduced the organic content ten-fold, which reduced or eliminated phytotoxic effects . Wheat and potato production studies were successful using effluents from reactors having with 8- to 1-day retention times . Aerobic bioreactor effluents supplied at least half of the crops elemental mass needs in these studies . Descriptions of leachate and effluent mineral content, biomass productivity, microbial activity, and nutrient budgets for potato and wheat are presented. Acta Astronaut, 1989 Apr, 19(4), 365 - 75 Analysis of an algae-based CELSS . Part 2: options and weight analysis; Holtzapple MT et al.; Life support components are evaluated for application to an idealized closed life support system which includes an algal reactor for food production . Weight-based trade studies are reported as "break-even" time for replacing food stores with a regenerative bioreactor . It is concluded that closure of the life support gases (oxygen recovery) depends on the carbon dioxide reduction chemistry and that an algae-based food production can provide an attractive alternative to re-supply for longer duration missions. Compost Sci Util, 1997 Summer, 5(3), 25 - 31 An overview: recycling nutrients from crop residues for space applications; Strayer RF et al.; Without some form of regenerative life support system, long duration space habitation or travel will be limited severely by the prohibitive costs of resupplying air, water, and food from Earth . Components under consideration for inclusion in a regenerative life support system are based on either physicochemical or biological processes . Physicochemical systems would use filtration and elemental phase changes to convert waste materials into usable products, while biological systems would use higher plants and bioreactors to supply crew needs . Neither a purely biological nor strictly a physicochemical approach can supply all crew needs, thus, the best each approach can offer will be combined into a hybrid regenerative life support system . Researchers at Kennedy Space Center (KSC) Advanced Life Support Breadboard Project have taken the lead on bioregenerative aspects of space life support . The major focus has been on utilization of higher plants for production of food, oxygen, and clean water . However, a key to any regenerative life support system is recycling and recovery of resources (wastes) . In keeping with the emphasis at KSC on bioregenerative systems and with the focus on plants, this paper focuses on research with biologically-based options for resource recovery from inedible crop residues. Sci Med (Phila), 1997 May-Jun, 4(3), 46 - 55 Tissue culture in microgravity; Duray PH et al.; Attempts to simulate normal tissue microenvironments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins . Gravity is one of the problems, tending to separate components that should be kept together . For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space. Gravit Space Biol Bull, 1998 May, 11(2), 31 - 9 Bioregenerative {correction of bioregnerative} life support: not a picnic; Knott WM; If humans are to live permanently in space, regenerative life support systems are an enabling technology and must replace the picnic approach of taking all supplies required for each mission . These systems are classified by technologies as either physical/chemical or bioregenerative . Both of these system-types can recycle water, remove carbon dioxide, produce oxygen, and recover essential elements from waste products . Bioregenerative can also produce food, thus, making it essential if humans are to exist in space independent of earth . A solely bioregenerative life support system includes plants as a biomass production module and microbial organisms in bioreactors as a resource recovery module . In the Advanced Life Support Program, bioregenerative life support systems are being investigated through a research and technology development project which includes large scale testing as part of the Breadboard Project and human tests conducted in the soon to be constructed BioPlex facility . Research and technology development efforts are directed toward optimizing biomass productivity in controlled chambers by developing light weight, energy efficient, and automated systems; recycling liquid and solid wastes; baselining the operation of bioreactors; determining system microbial stability; assessing chemical contamination; and building models required for long term system operations . The program will include space flight studies in the near future to determine if these life support technologies will function in microgravity . When a bioregenerative system is finally incorporated into a mission, the conversion from a picnic and resupply mentality to permanent recycling and independence from earth will be complete. Adv Space Res, 1994 Nov, 14(11), 75 - 8 Development of a CELSS bioreactor: oxygen transfer and micromixing in parabolic flight; Villeneuve PE et al.; The gas exchange portion of a phase-separated loop bioreactor was tested with respect to oxygen mass transfer and micromixing in accelerations of 0.01g, 1g, and 2g . A plot of the overall mass transfer coefficient versus gravity indicates the rate of oxygen transfer does not change as a function of acceleration . Also, it was determined that the micromixing did not exhibit significant changes in the various gravitational fields . These observations indicate the loop bioreactor should function independent of acceleration. Space Med Med Eng (Beijing), 1997 Feb, 10(1), 71 - 3 {Technological analysis of oxygenation of space bioreactor {correction of bioreacror}}; Tan Y; It is a difficult process to oxygenate the bioreactor . The microgravity environment brings more problems to this process . For example, gas bubbles will not rise through the fermentation media because of absence of buoyancy forces, so typical bioreactor on earth will not operate under microgravity . In this paper, the problem and possible solutions are discussed theoretically. Acta Astronaut, 1994 Jul, 33, 179 - 87 Effect of simple shear flow on photosynthesis rate and morphology of micro algae; Mitsuhashi S et al.; The convective motion of micro algal suspension gives an advantageous effect on the photosynthetic rate in the bioreactor, however, the nature of convective effect on the photosynthesis has not been fully understood . The purpose of this study concerns the nature of photosynthetic rate in a well-defined hydrodynamic shear flow of Spirulina platensis suspension, generated in a double rotating coaxial cylinders . The double rotating coaxial cylinders was installed in the incubator chamber with the controlled illumination intensity and temperature . Two kind of experiments, short and long term experiments, were performed to evaluate the direct effect of shear flow on the photosynthetic rate . The short term experiment indicates that the simple shear flow enables to augment the photosynthesis of Spirulina suspension and simultaneously causes the cell destruction due to the excessive shear stress . The long term experiment for 100 hours reveals that the growth rate and the morphology of Spirulina is sensitive to the external fluid mechanical stimulus . The long term application of mechanical stress on the algae may result in the adaptation of the photosynthetic function and morphology. Life Support Biosph Sci, 1996, 3(1-2), 67 - 74 Nitrogen dynamics in the CELSS Breadboard facility at Kennedy Space Center; Stutte GW; For the past 9 years, the Breadboard Project at Kennedy Space Center has studied the feasibility of using crop plants in bioregenerative life support systems for long-duration space missions . Nitrogen (N) has been emphasized in nutrient balance studies because it is a major plant nutrient, undergoes biogenic and abiogenic transformations, and is often limiting to plant growth under field conditions . Nitrogen budgets have been calculated from experimental results to quantify utilization and losses associated with specific crop production systems . The Breadboard Project has recently completed a 418-day potato crop study using recycled nutrient solution to evaluate the impact of continuous production on life support functions . A continuous production system is desirable in maintaining N balance within a solution because crop uptake rates vary dramatically depending upon the stage of crop development . Strategies for recycling N using biological techniques (e.g., biomass degradation with microbial bioreactors) have required that the production system be modified to distribute inputs more evenly over time . Recovery of N is dependent on the form of N entering the bioreactor and the desired output . Aerobic and anaerobic bioreactors for the recovery of N from waste streams and its transformation into a form usable by higher plants are being designed and tested. Adv Space Res, 1996, 18(1-2), 281 - 7 Comparison of aerobically-treated and untreated crop residue as a source of recycled nutrients in a recirculating hydroponic system; Mackowiak CL et al.; This study compared the growth of potato plants on nutrients recycled from inedible potato biomass . Plants were grown for 105 days in recirculating, thin-film hydroponic systems containing four separate nutrient solution treatments: (1) modified half-strength Hoagland's (control), 2) liquid effluent from a bioreactor containing inedible potato biomass, 3) filtered (0.2 micrometer) effluent, and 4) the water soluble fraction of inedible potato biomass (leachate) . Approximately 50% of the total nutrient requirement in treatments 2-4 were provided (recycled) from the potato biomass . Leachate had an inhibitory effect on leaf conductance, photosynthetic rate, and growth (50% reduction in plant height and 60% reduction in tuber yield) . Plants grown on bioreactor effluent (filtered or unfiltered) were similar to the control plants . These results indicated that rapidly degraded, water soluble organic material contained in the inedible biomass, i.e., material in leachate, brought about phytotoxicity in the hydroponic culture of potato . Recalcitrant, water soluble organic material accumulated in all nutrient recycling treatments (650% increase after 105 days), but no increase in rhizosphere microbial numbers was observed. Adv Space Res, 1996, 18(4-5), 139 - 48 New instrumentation for optical measuring of oxygen in gas or dissolved in liquids; Trettnak W et al.; The optical oxygen sensor is a novel device for the determination of oxygen in gases or dissolved in liquids . It is based on the measurement principle of fluorescence quenching, which is completely different from that of polarographic oxygen sensors (today the most widespread devices of oxygen detection) . The new instrument offers features and advantages, which render it not only a realistic alternative, but, for specific applications, make it superior to existing electrochemical methods . The system is based on low-cost semiconductor devices (light-emitting diodes, photodiodes, low-cost analogue and digital components) and new LED-compatible oxygen-sensitive membranes . The flow cell of the instrument may be thermostatted and the sensor can be calibrated by a simple two-point calibration procedure . The optical oxygen sensor is particularly suitable for measuring dissolved oxygen in respirometry, since no oxygen is consumed by the device and the signal is independent of sample flowrate or stirring speed . Typical fields of application are monitoring of oxygen in ground and drinking water, in process control in bioreactors and in breath gas and blood gas analysis. Physiologist, 1988, 31(1 Suppl), S52 - 5 Cell bioprocessing in space: applications of analytical cytology; Todd P et al.; Cell bioprocessing in space consists of the preparation, cultivation, purification and investigation of cells and their products in the microgravity environment of orbital space flight . Inertial acceleration is used as an independent variable to explore the limits of specific bioprocessing functions, such as cell growth and secretion, gravity-dependent phenomena in cell bioreactors, cell fusion, the influence of thermal convection on processes at cellular dimensions, the electrophoretic separation of cell subpopulations and subcellular particles, and two-phase partitioning of cells, bioparticles, and macromolecules . Analytical cytology techniques are under development for on-orbit application to future cell growth and separation experiments, such as those anticipated in the Space Station era. Appl Micrograv Technol, 1988, 3, 115 - 22 Cultivation of single cells in space; Gmunder FK et al.; The purpose of this review is to present an updated and comprehensive analysis of the experiments with single cells performed in space . Especially the results of the investigations performed in Biorack on the D-1 mission clearly show that important cellular functions are changing in microgravity . Cell proliferation, differentiation, metabolism, membrane properties, and cytoplasmic streaming underwent significant alteration during exposure to space flight conditions in a variety of single cells cultures spanning from bacteria to mammalian cells . These findings open new and interesting perspectives to basic and applied research in microgravity . The focus of this paper is on the cultivation of mammalian cells in space laboratories and on the related instrumentation . While Biorack is a useful and efficient instrument for simple studies in Spacelab, the development of new facilities like incubators with automated fixation devices as well as of more complex bioreactors is strongly recommended. Waste Manag Res, 1991 Oct, 9(5), 471 - 9 Performance characteristics of a low sludge bioreactor for wastewater treatment; Petrie GE et al.; An immobilized microbial cell system was developed and tested for the treatment of industrial wastewater . A consortium of selected aerobic microorganisms was immobilized onto several different support matrices in a packed bed reactor configuration and operated in a continuous process mode . Comparison of the support matrices showed only small differences in treatment efficiency, but significant differences in sludge production and process stability . Porous polymer supports were highly resistant to feedstream upsets and produced 80% lower sludge solids as compared with non-porous supports . These results were seen at both the benchtop and pilot plant scale for treatment of complex industrial waste streams . This technology was applied, in preliminary experiments, to the treatment of a model waste stream simulating wastewater from a Controlled Ecological Life-Support System (CELSS). Adv Space Res, 1989, 9(8), 185 - 93 Phase separated membrane bioreactor: results from model system studies; Petersen GR et al.; The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described . The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out . Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls . Methods of upgrading the design to eliminate these resistances are proposed . Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestrial {correction of terrestial} simulation. Adv Space Res, 1989, 9(11), 119 - 27 Mammalian cell cultivation in space; Gmunder FK et al.; Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors . Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material . However, expertise for this venture is not at hand . A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed . It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system . The cell chambers have a volume of 200 microliters . Medium exchange is achieved with an automatic osmotic pump . The system is neither mechanically stirred nor equipped with sensors . Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers . The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion . Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS . On ground tests suggest that this system is feasible. Adv Space Res, 1987, 7(4), 47 - 52 Sunlight supply and gas exchange systems in the microalgal bioreactor; Mori K et al.; The bioreactor with sunlight supply system and gas exchange systems presented here has proved feasible in ground tests and shows much promise for space use as a CELSS device . Our chief conclusions concerning the specification of total system needed for a life support system for a man in a space station are the following . (1) Sunlight supply system: compactness and low electrical consumption . (2) Bioreactor system: high density and growth rate of chlorella . (3) Gas exchange system: enough for O2 production and CO2 assimilation. Adv Space Res, 1992, 12(5), 237 - 45 Evolution of a phase separated gravity independent bioreactor; Villeneuve PE et al.; The evolution of a phase-separated gravity-independent bioreactor is described . The initial prototype, a zero head-space manifold silicone membrane based reactor, maintained large diffusional resistances . Obtaining oxygen transfer rates needed to support carbon-recycling aerobic microbes is impossible if large resistances are maintained . Next generation designs (Mark I and II) mimic heat exchanger design to promote turbulence at the tubing-liquid interface, thereby reducing liquid and gas side diffusional resistances . While oxygen transfer rates increased by a factor of ten, liquid channeling prevented further increases . To overcome these problems, a Mark III reactor was developed which maintains inverted phases, i.e., media flows inside the silicone tubing, oxygen gas is applied external to the tubing . This enhances design through changes in gas side driving force concentration and liquid side turbulence levels . Combining an applied external pressure of four atmospheres with increased Reynolds numbers resulted in oxygen transfer intensities of 232 mmol O2/l/h (1000 times greater than first prototype and comparable to a conventional fermenter) . A 1.0 liter Mark III reactor can potentially deliver oxygen supplies necessary to support cell cultures needed to recycle a 10 astronaut carbon load continuously. J Biotechnol, 1999 Apr 30, 70(1-3), 289 - 97 An integrated solar and artificial light system for internal illumination of photobioreactors; Ogbonna JC et al.; Exploitation of photosynthetic cells for the production of useful metabolites requires efficient photobioreactors . Many laboratory scale photobioreactors have been reported but most of them are extremely difficult to scale up . Furthermore, the use of open ponds and outdoor tubular photobioreactors is limited by the requirement for large spaces and the difficulty in maintaining sterile conditions . In view of this, we have designed and constructed an internally illuminated stirred tank photobioreactor . The photobioreactor is simple, heat sterilizable and mechanically agitated like the conventional stirred tank bioreactors . Furthermore, it can easily be scaled up while maintaining the light supply coefficient and thus the productivity constant . A device was installed for collecting solar light and distributing it inside the reactor through optical fibers . It was equipped with a light tracking sensor so that the lenses rotate with the position of the sun . This makes it possible to use solar light for photosynthetic cell cultivation in indoor photobioreactors . As a solution to the problems of night biomass loss and low productivity on cloudy days, an artificial light source was coupled with the solar light collecting device . A light intensity sensor monitors the solar light intensity and the artificial light is automatically switched on or off, depending on the solar light intensity . In this way, continuous light supply to the reactor is achieved by using solar light during sunny period, and artificial light at night and on cloudy days. Nat Med . 1997 Mar;3(3):259. Cells in space Wong J. How does one treat in a seriously injured astronaut in outer space or even another planet? To answer such a question, the US National Aeronautical Space Administration (NASA) has embarked on a program of growing tissues--and possibly whole organs--in space . NASA has developed a unique rotating bioreactor that allow cells to be grown in a microgravity environment that eliminates almost all shear forces placed upon a cell culture system while entering space . Back on earth, this novel bioreactor has led to exciting discoveries and applications by scientists trying to get cells to differentiate and form their natural three-dimensional tissue matrices--the holy grail of tissue engineers . NASA's bioreactor has allowed various labs to culture cells and even viruses previously impossible to grow using traditional methods . These successes are attributed to the bioreactor's ability to provide an unique environment that closely resembles tissue differentiation during embryogenesis, and thus allowing cellular expression of surface epitopes similar to that of intact tissues . It also appears that cells grown in a microgravity, low-shear environment allows for greater chemical signaling, probably as a result of more surface contact between cells . Realizing the bioreactor's commercial potential, Santa Monica, California-based VivoRx licensed exclusive rights from NASA for both therapeutic and diagnostic commercial applications . VivoRx has, in the past, successfully transplanted encapsulated islet cells from cadavers and porcine pancreas into insulin-dependent diabetics, perhaps a major breakthrough in the treatment of diabetes . However, pancreas from cadavers are in very short supply . The bioreactor may be the answer; VivoRx hopes the bioreactor will allow them to propagate enough human islet cells to use their cell-based approach to treat a large diabetic population . The company has already successfully grown islet cells generated from the bioreactors, and is beginning FDA-approved Phase I/II clinical trials. Biotechnol Bioeng, 2001 Oct 20, 75(2), 204 - 11 Use of the chemiluminescent probe lucigenin to monitor the production of the superoxide anion radical in a recombinant Aspergillus niger (B1-D); Bai Z et al.; Direct detection of intracellular superoxide anion radical (O(2)(.-)) production is of critical importance for investigating the responses of filamentous fungi to oxidative stress in bioprocesses . The purpose of this study is to establish a reliable method to monitor the O(2)(.-) production within pellets of Aspergillus niger . Addition of pure oxygen and the redox cycling agent paraquat to fungal pellet suspensions resulted in a considerable increase in lucigenin-derived chemiluminescence (LDCL) . In the presence of exogenous superoxide dismutase (SOD), the LDCL of a disrupted cell solution was inhibited . In contrast, with addition of diethyldithiocarbamate and sodium azide, respectively, the inhibitors of Cu, Zn-SOD and Mn-SOD, an increased LDCL was observed . Further, as a probe, lucigenin can be absorbed and accumulated in fungal pellet within a few minutes . Various pretreatments of the bioreactor sample for the measurement of LDCL, were also investigated in the present study, and the use of intact pellets was adopted here rather than disrupting cells because the latter treatment led to difficulties in LDCL measurement . These results show that lucigenin may be used as a convenient chemiluminescent probe to monitor intracellular production of O(2)(.-) in filamentous fungi, and thus to follow changes in the level of this stressor within fungi Biotechnol Bioeng, 2001 Oct 20, 75(2), 197 - 203 Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells; Meissner P et al.; Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins . We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume . Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD) . Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA) . Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100% . Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest . The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L . An E . coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L . The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell . Appl Environ Microbiol, 2001 Sep, 67(9), 4374 - 6 Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples; Singleton DR et al.; To determine the significance of differences between clonal libraries of environmental rRNA gene sequences, differences between homologous coverage curves, CX(D), and heterologous coverage curves, CXY(D), were calculated by a Cramer-von Mises-type statistic and compared by a Monte Carlo test procedure . This method successfully distinguished rRNA gene sequence libraries from soil and bioreactors and correctly failed to find differences between libraries of the same composition. J Biotechnol, 2001 Sep 13, 91(1), 75 - 81 Docosahexaenoic acid ethyl esters from Isochrysis galbana; Poisson L et al.; In order to produce docosahexaenoic acid (DHA), a culture of the microalgal strain Isochrysis galbana was implemented . In Erlenmeyer flasks, a natural seawater medium, the Provasoli 1/3 medium, was compared to the classical Jones medium for DHA production . The Provasoli 1/3 medium stimulated growth (0.44 d(-1)), but influenced DHA accumulation negatively (0.240 pg cell(-1)) . However, DHA production per liter of culture medium were of the same order of magnitude with both media (0.961 mg l(-1)) . In a 2-l bioreactor, DHA production per liter of culture medium did not increase significantly between 4 and 8 days of culture . With a view to optimize DHA productivity, cells should be harvested at the end of exponential phase i.e . after 4 days of culture . Two strategies were then attempted to produce DHA ethyl esters . First, lipids from I . galbana were submitted to lipase-catalyzed transesterification with ethanol . Secondly, fatty acids from I . galbana were submitted to lipase catalyzed esterification with ethanol . In both cases, lipase from Candida antarctica was shown to be the best candidate, among the five tested, with conversion yields of 20 and 60% after 24 h of transesterification and esterification respectively. J Microencapsul, 2001 Sep-Oct, 18(5), 567 - 76 New strategy for the cultivation of microalgae using microencapsulation; Joo DS et al.; The four species of microalgae (Dunaliella bardawil, Chlorella minutissima, Pavlova lutheri and Haematococcus pluvialis) were immobilized in Ca-alginate capsules as a basic study for the development of the economic cultivation process . Under the batch culture of aerobic conditions, the thickness of the capsule membrane and CO2 supply did not affect the growth of the immobilized microalgae, Dunaliella bardawil . Cell concentration of immobilized microalgae in the capsule was higher than those of immobilized microalgae in beads and free cells . The cell concentrations of microencapsulated Dunaliella bardawil and Haematococcus pluvialis were five times greater than that of free cells . Based on these results, microencapsulation for the culture of microalgae was an effective method for the high-density cultivation . In comparison to the immobilized cultivation on the bioreactor type, it was more effective for the cultivation in the bubble column bioreactor than that in the stirrer tank bioreactor. Biotechnol Bioeng, 2001 Sep, 76(2), 115 - 25 Longterm stability of phase I and phase II enzymes of porcine liver cells in flat membrane bioreactors; Langsch A et al.; Recently, researchers have focused on the use of bioartificial liver devices to support patients with fulminant hepatic failure . Our team developed a cell-based flat membrane bioreactor (FMB) . In this, porcine liver cells were maintained in 3D-coculture between two gel layers in a sandwich configuration for 3 weeks to study the influence of this bioreactor technique on the preservation of basic, not induced activities of phase I and phase II enzymes . First, the time and substrate dependencies of the following enzymes were measured: ethoxyresorufin-O-deethylase (EROD, CYP 1A1/1A2) and ethoxycoumarin-O-deethylase (ECOD, CYP 2B6) as phase I enzymes, and glutathione-S-transferase (GST), UDP-glucuronosyltransferase (UGT) and sulfotransferase (ST) as phase II enzymes . To find optimal test conditions Michaelis-Menten kinetics were calculated . Next, different potential inducers were tested to find out the most effective compounds . Based on these results, the basic, not induced levels of the different enzymes were determined in the flat membrane bioreactor . Furthermore, the response of these enzyme activities to the chosen inducers was investigated to examine whether the cells keep their ability for drug-drug interactions . Basic, not induced activities of both phase I enzymes and the phase II enzymes GST and UGT were maintained at nearly the initial levels during the complete period of study . In addition, it was possible to induce these enzymes twice or three times in a weekly interval . In contrast, the basic, not induced activity of ST increased during the first 10 days of culture . It stabilized then and was maintained steady . As in short-term investigations, no reaction of the ST-activity towards any inducer could be obtained . These results prove that porcine liver cells preserve their phase I and phase II activities and respond to inducing drugs over 3 weeks in culture . Therefore, the flat membrane bioreactor is not only suitable for investigating drug metabolism, drug-drug interactions, and enzyme induction but also for supporting liver functions . Cent Eur J Public Health, 2001 May, 9(2), 102 - 5 Potential application of immobilized and perfused hepatocytes in environmental toxicology studies; Farghali H et al.; Conventional cellular models have contributed significantly to the understanding of many aspects of cell physiology and molecular biology . In these models cells are metabolically less active, due to the inefficient oxygenation and waste product buildup . Therefore perfusion methods for the cells are expected to improve cell activities . Cells have to be fixed in or on an appropriate inert carrier or support, which enables cellular perfusion, maintains integrated cellular functions and makes a bioreactor . Since isolated hepatocytes are extensively used in biomedical studies including those dealing with environmental pollutants or toxins and in xenobiotic biotransformation investigations, an efficient hepatocyte perfusion model has to be available for researchers . This research article is focusing on the value of hepatocyte immobilization as a laboratory bioreactor model and is shedding light on its potentiality in research related to public health . We demonstrate the application of this cellular model as a means to study representative phase I and phase II biotransformation reactions using hexobarbital hydroxylation and 7-ethoxycoumarin deethylation and 4-chloro 2-dinitrobenzene glutathione . Both phase I and phase II drug biotransformation in hepatocytes was demonstrated in this study non-destructively to the cells and in an efficient way . In spite of the aforementioned advantages, immobilized hepatocytes yet have relatively limited applications compared to conventional hepatocyte cellular systems . Reasons for this discrepancy are discussed . This cellular system may become popular due to the better performance of immobilized hepatocytes as compared to conventional hepatocyte culture and due to economic and ethical reasons . Naturally its applicability will cover several biomedical areas including basic research in environmental toxicology and other public health issues. J Biotechnol, 2001 Aug 23, 89(2-3), 185 - 92 Integration of biotechnological wastewater treatment units in textile finishing factories: from end of the pipe solutions to combined production and wastewater treatment units; Feitkenhauer H et al.; Increasing costs for water, wastewater and energy put pressure on textile finishing plants to increase the efficiency of wet processing . An improved water management can decrease the use of these resources and is a prerequisite for the integration of an efficient, anaerobic on-site pretreatment of effluents that will further cut wastewater costs . A two-phase anaerobic treatment is proposed, and successful laboratory experiments with model effluents from the cotton finishing industry are reported . The chemical oxygen demand of this wastewater was reduced by over 88% at retention times of 1 day or longer . The next step to boost the efficiency is to combine the production and wastewater treatment . The example of cotton fabric desizing (removing size from the fabric) illustrates how this final step of integration uses the acidic phase bioreactor as a part of the production and allows to close the water cycle of the system. J Biotechnol, 2001 Aug 23, 89(2-3), 99 - 106 A packed-bed fungal bioreactor for the continuous decolourisation of azo-dyes (Orange II); Mielgo I et al.; The degradation of an azo dye, Orange II, by immobilised Phanerochaete chrysosporium in a continuous packed bed bioreactor for periods longer than 30 days has been carried out . Nearly complete decolourisation (>95%) was achieved when working at a high dye load rate of 0.2 g x l(-1) x d(-1), a temperature of 37 degrees C, a hydraulic retention time (HRT) of 24 h and applying oxygen gas in a pulsed flow . These conditions allowed Manganese peroxidase (MnP) production and the subsequently Orange II decolourisation . A correlation between residual MnP activity in the effluent and decolourisation was established . Apparently, for decolourisation to be effective, a minimum MnP activity was required, no substantial increase in efficiency at MnP activities higher than 10 U x 1(-1) was observed . The treatment caused, the breakdown of the chromophoric group as well as the cleavage of the aromatic ring. Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 276 - 9 Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli; Deng X et al.; Genetically engineered E . coli, which express both a Hg2+ transport system and metallothionein, were tested for their ability to remove mercury from wastewater . The wastewater contained more than ten different ions, including 2.58 mg/l mercury, and its pH was 9.6 . Mercury uptake was faster from the wastewater than from distilled water, probably because of the higher ionic strength, as the high pH had little effect on mercury accumulation . EDTA also stimulated mercury uptake rather than inhibiting it . A hollow-fiber bioreactor was used to retain induced cells for continuous mercury uptake . The cells removed more than 99% of the mercury in the wastewater and the final amount of mercury accumulated was 26.8 mg/g cell dry weight, while none of the other ions were removed from the water . These results indicated that the induced cells had a high affinity and specificity for mercury. Anasthesiol Intensivmed Notfallmed Schmerzther, 2001 Jul, 36(7), 440 - 4 {Genetically engineered drugs and their application with the example of erythropoietin}; Donatz V et al.; Over the past two decades, many genetically engineered drugs have been developed and approved for the treatment of patients . Typically, these drugs are characterized by a high and specific activity in the presence of optimal safety . They include hormones, enzymes, growth and coagulation factors, antibodies as well as vaccines . All these proteins are generated using recombinant DNA technology . An expression vector with the gene encoding for the protein of interest is introduced into an appropriate microorganism or cell line . The biochemical machinery of the host cell then translates the genetic information into the corresponding protein . Large scale production of the recombinant drugs uses biotechnological processes . The genetically modified organisms are grown in bioreactors from which the desired protein is finally isolated and purified . This review focuses on the production and clinical application of recombinant erythropoietin in the areas of nephrology, hemato-oncology and elective surgery. Rev Argent Microbiol, 2001 Apr-Jun, 33(2), 59 - 64 {Degradation of plant waste by Coprinus truncorum using 2 culture methods}; Diorio LA et al.; Degradation of yard wastes by Coprinus truncorum growing in a vertical aereated bioreactor or in flasks was studied . There was a constant decay of reducing sugars in the medium that avoided their accumulation and their possible repression of degradative enzymes . Endoxylanase activity at first showed a similar pattern in both culture conditions, with maximal activity on the 12th day, but flasks maintained a high activity thereafter . Flasks also showed a higher endoglucanase activity with a peak on the 18th day, whereas the maximal value in the bioreactor was reached on the 26th day . No Mn-peroxidase and only low values of laccase activity were found . The measurements of pH and soluble proteins during the incubation period were suitable indicators of the degradation process by C . truncorum. Artif Organs, 2001 Jul, 25(7), 571 - 8 In vitro and in vivo evaluation of albumin synthesis rate of porcine hepatocytes in a flat-plate bioreactor; Shito M et al.; Several configurations of extracorporeal bioartificial liver devices have been developed for the potential treatment of fulminant hepatic failure or as a bridge to liver transplantation . Recently, we developed a microchannel flat-plate bioreactor with an internal membrane oxygenator in which porcine hepatocytes are cultured as a monolayer on the bottom glass surface . In the present study, we investigated synthetic function of porcine hepatocytes in the bioreactor in both in vitro and in vivo flow circuit models . In vitro, albumin synthesis was stable in the bioreactor for up to 4 days of perfusion . In vivo, with the extracorporeal connection of the bioreactor to rat vasculature, porcine albumin was detectable for 24 h in the rat plasma . We also developed a simple mathematical model to predict the in vivo porcine albumin concentration in rat plasma . These results indicate that this configuration of a microchannel flat-plate bioreactor has potential as a liver support device and warrants further investigation. Artif Organs, 2001 Jul, 25(7), 566 - 70 Bioartificial liver treatment prolongs survival and lowers intracranial pressure in pigs with fulminant hepatic failure; Khalili TM et al.; Intracranial hypertension leading to brainstem coning is a major cause of death in fulminant hepatic failure (FHF) . We have developed a bioartificial liver (BAL) utilizing plasma perfusion through a bioreactor loaded with porcine hepatocytes and a column with activated charcoal . In a Phase I clinical trial, we observed a decrease in intracranial pressure (ICP) in FHF patients . However, these patients received BAL therapy together with other measures . We therefore examined whether BAL therapy alone could prevent development of intracranial hypertension in pigs with surgically induced FHF . Pigs (40-60 kg) underwent end-to-side portacaval shunt, transection of all hepatic ligaments, and placement of slings around the hepatic artery and bile duct . After 3 days, the slings were tightened to induce liver necrosis . After 4 h, Group 1 pigs (n = 6) underwent a 6 h treatment with the BAL utilizing 10 billion cryopreserved pig hepatocytes and a charcoal column, Group 2 pigs (n = 6) with the BAL containing charcoal but no cells, and Group 3 pigs (n = 6) with the BAL containing neither cells nor charcoal . Group 1 pigs maintained a normal ICP during BAL treatment and for 14 h afterward and because of this effect they survived longer than Groups 2 and 3 animals . In contrast, Groups 2 and 3 pigs showed an early (6-8 h) rise in ICP. Biodegradation, 2000, 11(5), 295 - 303 Use of hydrophobic membranes to supply hydrogen to sulphate reducing bioreactors; Fedorovich V et al.; This paper reports on the application of hydrophobic membranes to supply the gaseous substrates hydrogen/carbon dioxide (H2/CO2) to a sulphate reducing bioreactor . For this, two flat 0.016 m2 sheets of flouroplast microporous (0.45 microm) membranes were inserted in a 3.6 dm3 bioreactor for the supply of H2/CO2 gas as small gas bubbles . The bioreactor was operated at 30 degrees C and pH 7.0 and was also equipped with an external ultra filtration module for biomass retention . At a sulphate loading rate (SLR) of 1.32 g SO4(2-) dm(-3) day(-1) and a hydraulic retention time (HRT) of 61 h, a sulphate reduction rate (SRR) of 0.90 g SO4(2-) dm(-3) day(-1) was achieved . When the influent sulphate concentration was reduced from 3.36 to 0.75 g SO4(2-) dm(-3) by lowering the HRT to 10.3 h (SLR of 1.75 g SO4(2-) dm(-3) day(-1) the SRR dropped to 0.22 g SO4(2-) dm(-3) day(-1) . The lower sulphate reduction efficiency was most probably caused by a too short biomass-substrate contact time or by irreversible sulphide inhibition . Mass transfer limitation of H2 and improper mixing of the reactor liquid were shown not to contribute to the low sulphate reduction efficiency. Biotechnol Prog, 2001 Jul-Aug, 17(4), 661 - 3 Hairy roots of Brugmansia candida that grow without agitation: biotechnological implications; Pitta-Alvarez SI et al.; Hairy roots of Brugmansia candida that grew without agitation were obtained . Kinetics of growth and production of the tropane alkaloids scopolamine and hyoscyamine, with and without agitation, were studied . The exponential growth rate was higher in the roots that were exposed to shaking (0.13 d(-1)) than in the nonagitated ones (0.09 d(-1)) . The specific production and the levels per flask of both alkaloids were enhanced without shaking . The use of these roots in large-scale productions could be economically advantageous . It remains to be seen if the data obtained in shake flasks can be extrapolated to large-scale bioreactors. Biotechnol Prog, 2001 Jul-Aug, 17(4), 647 - 60 Bifurcation analysis of continuous biochemical reactor models; Zhang Y et al.; The validity of a biochemical reactor model often is evaluated by comparing transient responses to experimental data . Dynamic simulation can be a rather inefficient and ineffective tool for analyzing bioreactor models that exhibit complex nonlinear behavior . Bifurcation analysis is a powerful tool for obtaining a more efficient and complete characterization of the model behavior . To illustrate the power of bifurcation analysis, the steady-state and transient behavior of three continuous bioreactor models consisting of a small number of ordinary differential equations are investigated . Several important features, as well as potential limitations, that are difficult to ascertain via dynamic simulation are disclosed through the bifurcation analysis . The results motivate the use of dynamic simulation and bifurcation analysis as complementary tools for analyzing the nonlinear behavior of bioreactor models. Biotechnol Prog, 2001 Jul-Aug, 17(4), 634 - 42 Identification and control of dissolved oxygen in hybridoma cell culture in a shear sensitive environment; Simon L et al.; The productivity of mammalian cells can be enhanced by facilitating adequate oxygen transfer into the cultivation medium . However, current methods of controlling dissolved oxygen (DO) fail to account for alterations in medium composition during the course of the fermentation . These changes, which directly affect gas solubility and overall mass transfer coefficient, may be significant and deteriorate controller's performance in the long run . In this paper, the applications of Generalized Predictive Controllers (GPC) to DO control were investigated in a shear sensitive environment and compared to PID and Model Predictive Controllers (MPC) . Input and output data for system identification were initially generated by varying the composition of oxygen fed into the bioreactor from 0 to 0.21 mol % while keeping the total inlet gas flow rate at 8.75 vvm . The process was identified using an AutoRegressive model with eXogeneous inputs (ARX) model and tested on different data sets . The model parameters were then correlated with the overall mass transfer coefficients . In simulation tests, the output of the PID controller switched from minimum to maximum values while more continuous control signals were obtained with the MPC and GPC controllers . When tested in a cell-free medium, all three controllers were able to track setpoint changes with some chattering observed in the control signals . The GPC outperformed the MPC and PID controllers when applied to the cultivation of hybridoma cells. Biotechnol Prog, 2001 Jul-Aug, 17(4), 606 - 11 Effects of in situ cobalt ion addition on the activity of a gfp-oph fusion protein: the fermentation kinetics; Wu CF et al.; The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E . coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein . It was found that cobalt ion addition improved the OPH activity significantly . When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts . Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity . Also, the maximum OPH activity was not linearly related to inducer concentration . A mathematical model was then constructed to simulate these phenomena . Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity . Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor. Biotechnol Prog, 2001 Jul-Aug, 17(4), 597 - 605 Classification of static behavior of a class of unstructured models of continuous bioprocesses; Ajbar A; The stability characteristics of a class of unstructured models of continuous bioreactors are analyzed using elementary concepts of singularity theory and continuation techniques . The class consists of models for which the non-biomass product formation rate is linearly proportional to the utilization rate of limiting substrate . The kinetics expressions of cell growth and product synthesis are allowed to assume general forms of substrate and product . Global analytical conditions are derived that allow the construction of a practical picture in the multidimensional parameter space delineating the different static behavior these models can predict, including unique steady states, coexistence of non-trivial steady states with wash-out conditions, and multistability resulting from hysteresis . These general results are applied to specific examples of bioprocesses and allow the study of the effect of kinetic and operating parameters on the stability characteristics of these models. Protein Expr Purif, 2001 Aug, 22(3), 414 - 21 Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization; Wolff MW et al.; C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway . Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal . C1INH replacement therapy may be lifesaving for patients with this disorder . The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH) . A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter . Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg . Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure . This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH . Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH . The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis . Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH . Biochim Biophys Acta, 2001 Aug 15, 1527(3), 112 - 22 The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger; Wallis GL et al.; The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied . We have shown that a chan