Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

Engineering the Escherichia coli outer membrane protein OmpC for metal bioadsorption

The outer membrane protein, OmpC, from Escherichia coli was used to display metal-binding poly-histidine peptides on the surface of this bacterium. SDS-PAGE analysis of outer membrane protein preparations confirmed the expression of the metal-binding epitopes inserted in position 162 of the mature OmpC protein. Display of these epitopes was confirmed by epifluorescence microscopy of cells bound to Ni²⁺-NTA-agarose beads and metal adsorption experiments. The cells harboring one or two copies of the metal binding epitope were able to adsorb 3 to 6 times more Zn²⁺ (13.8 mol g^–1 cell), Fe³⁺ (35.3 mol g^–1 cell), and Ni²⁺ (9.9 mol g^–1 cell) metallic ions than control cells expressing the wild-type OmpC.     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

Plant development in long-term embryogenic callus lines of Cucurbita pepo

Embryogenic callus derived from pumpkin hypocotyl segments was induced and maintained for 15 years on MS medium supplemented with the auxins IBA (4.9 M), 2, 4-D (4.5 M) or IAA (5.7 M). On induction media continued embryo maturation and development of adult plants typically failed. Therefore, small embryogenic clumps and individually isolated embryos were subcultured two to four times on one of the conversion media: MS supplemented with 1.5% sucrose and (a) no hormone, (b) 2.9 M IAA, (c) 5.7 M IAA, (d) 11.4 M IAA, (e) 12 M IEt, (f) 3.8 M ABA or (g) 2% activated charcoal. The cell line and the kind of auxin used in the induction and maintenance medium, both had a marked influence on the development of plantlets. The best result was achieved with a line that has been induced and maintained for 15 years on MS with IBA. In the IBA line, out of 100 embryos, 77 developed into plantlets on MS medium supplemented with 11.4 M IAA.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid - 2, 4-D 2, 4-di-chlorophenoxyacetic acid - IEt indole-3-etha-nol - MS Murashige and Skoog (1962) medium     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

Effects of polymyxin B on the sarcoplasmic reticulum membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca²⁺-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 M, 130M and 550 M of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca²⁺-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 M polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.     

Screening for antiproliferative effects of cellular components from lactic acid bacteria against human cancer cell lines

Whole cells, cytoplasms and peptidoglycans of ten different lactic acid bacteria (LAB) were tested for in vitro cytotoxicity on diverse cancer cell lines using the ³H-thymidine incorporation assay. The peptidoglycans and cytoplasm fractions, as well as heat-killed whole cells of LAB, had significant antiproliferative activities against several cancer cell lines. In particular, the cytoplasm fractions exhibited marked direct antiproliferative activities against colon and gastric cancer cell lines, whereas the peptidoglycans retarded growth of colon and bladder cancer cell lines. The cytoplasm fractions of Bifidobacterium longum and Lactococcus lactis ssp. lactis inhibited proliferation of two cancer cell lines by 50% at 33 and 23 g ml^–1 for SNUC2A (a human colon adenocarcinoma cell line) and 17 and 11 g ml^–1 for SNU-1 (a human gastric cancer cell line), respectively.     

Transformation of 3β-(2-Hydroxyethoxy)-5α-cholest-8(14)-en-15-one to a Polar Metabolite in Hepatoma Hep G2 Cells

Incubation of 3-(2-hydroxy-2[³H]-ethoxy)-5-cholest-8(14)-en-15-one with Hep G2 cells led to the accumulation of a radioactive polar product in the culture medium, which was identified as 3-(2-hydroxyethoxy)-15-keto-5-cholest-8(14)-ene-24-oic acid. Its structure was confirmed by a chemical counter synthesis. The labeled ketosterol was rapidly (t _1/2 = 6 min) and reversibly bound by Hep G2 cells. The intracellular concentration of 15-ketosterol decreased during incubation mainly due to the formation of a polar metabolite, secreted to the medium. The level of cholesterol biosynthesis was 22 ± 5% of the control value in Hep G2 cells at a 15-ketocholesterol concentration in the medium of 30 M. However, further incubation for 3 h in the medium without the ketosterol led to restoration of the level of biosynthesis to 85 ± 11% of the control value. These results suggest that inhibition of the cholesterol biosynthesis by 15-ketocholesterol in Hep G2 cells depends on the intracellular concentration of the inhibitor, which, in turn, is determined by the rate of its conversion into the polar metabolite.     

Aluminium impacts elements of the phosphoinositide signalling pathway in neuroblastoma cells

Inositol phosphate formation was examined in aluminium-treated murine neuroblastoma cells labelled with [³H]-myoinositol. Employing fluoride-stimulated intact cells, aluminium (0.2M to 1 mM) reduced inositol phosphate formation in a dose-dependent manner. In digitonin-permeabilized cells, stimulated with nonhydrolyzable GTP[S], inositol phosphate formation was also inhibited by increasing aluminium doses; the IC₅₀ value was about 20M aluminium, while the inositol phosphate level was reduced 2.5 to 3 fold by 50M aluminium. The inhibitory effect of aluminium (50M) could not be reversed by increasing GTP[S] concentrations up to 500M. Prechelation of aluminium to citrate or EGTA completely abolished the aluminium-triggered inhibition of fluoride-stimulated inositol phosphate formation in intact cells, but had little effect on the inhibition of permeabilized cells stimulated with GTP[S]. In neuroblastoma cells phosphoinositide hydrolysis could be evoked either through a pathway involving the Mg²⁺/guanine nucleotide binding (G_p) protein, or via a pathway operative in the presence of high intracellular Ca²⁺ concentrations. In the Mg²⁺/G_p protein-mediated pathway, formation of inositol triphosphate, IP₃, inositol diphosphate, IP₂, and inositol monophosphate, IP, was apparently inhibited by aluminium in an interdependent manner. As to the Ca²⁺-mediated pathway, aluminium application mainly diminished the release of IP₃. Following interiorization, aluminium thus acts upon elements critical for phosphoinositide-associated signal transduction. An aluminium target apparently resides on the G_p protein. Phosphatidylinositol-4,5-diphosphate-specific phospholipase C probably harbours a second aluminium target.     

Ecotoxicity of lead to the zebra musselDreissena Polymorpha,Pallas

The effect of lead on the filtration rate of the zebra musselDreissena polymorpha was investigated, together with the accumulation of Pb in the soft tissues of the mussels. The NOEC-filtration was 116 g.l^–1 (0,56 mol.l^–1) and the EC₅₀-filtration was 370 g.l^–1 (1.79 mol.l^–1). The NOEC-accumulation was the concentration found in the control water (1.4g.l^–1). These experiments show that the EC₅₀-filtration for Pb is similar to that for Cd, higher than that for Cu and lower than that for Zn. The water quality criteria for lead allow 25 g Pb.l^–1 in surface water. This will not cause short-term effects. Long-term effects may, however, occur, since an accumulation of Pb as low as 16 g.l^–1 was recorded in this study.     

Effect of zearalenone (F-2) on pea stem,maize root,and rat liver mitochondria

At 5 and 10 g ml^-1 concentration, zearalenone (F-2), a mycotoxin produced by a number of species of the genus Fusarium, causes an inhibition of the oxidative phosphorylation of isolated plant mitochondria, while at 20 and 40 g ml^-1 it causes uncoupling. However, when the mitochondria are pre-incubated for 20 min with F-2, the uncoupling appears to be the prevailing effect. F-2 is also able to inhibit the mitochondrial ATPase activity (Mg²⁺-dependent). Conversely, F-2 (40 g ml^-1) does not alter the ATP level of maize roots and only slightly affects the ATPase activity of pea stem and maize root microsomal fractions. In addition, F-2 (10–40 g ml^-1) inhibits ATP synthesis catalyzed by rat liver mitochondria. It is suggested that the phytotoxicity of F-2, also known for its ability to collapse the transmembrane electric potential of maize roots, may be mainly linked to its ability to increase the proton permeability of the cell, similar to the common uncouplers.Abbreviations F-2 zearalenone - DCCD N,N-dicyclohexylcarbodiimide - PCCP carbonyl cyanide, p-trifluoromethoxiphenylhydrazone - CBT Cerospora beticola toxin     

Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells     

Cell characteristics and biochemical composition ofDunaliella primolecta Butcher conditioned at different concentrations of dissolved nitrogen

The rates of growth, cell size, elemental and biochemical composition ofDunaliella primolecta were monitored during exponential growth after conditioning over three weeks in media containing different concentrations of nitrogen. The rate of growth, measured both as cell density and cell volume, was correlated positively with the N concentration of the medium (P<0.01). N-starved cells did not grow and died within three days. Cell volume and dry weight per cell were significantly higher (P<0.01) in the N-low (0.45 g-at 1^–1 NO₃-N) condition than in the N-high I condition (3.53 g-at 1^–1 NO₃-N). In the N-high II condition the addition of 1.87 g-at 1^–1 NH₄-N to 3.53 g-at 1^–1 NO₃-N did not significantly enhance growth.The composition was influenced by the availability of nitrogen. The amount of protein per cell decreased significantly from 20.1 to 9.7 pg with reduced nitrate availability between the N-high condition and the N-low condition (P<0.01). The associated carbohydrate content increased significantly from 10.6 to 26.74 pg per cell (P<0.001). These findings confirm the potential for significant variability in the composition ofD. primolecta, both in the natural environment and following experimental manipulation.     

An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

In this work, metabolite and antibody production kinetics of hybridoma cultures were investigated as a function of cell density and growth rate in a homogeneous perfusion reactor. Hydrophilized hollow fiber polypropylene membranes with a pore size of 0.2 m were used for medium perfusion. Oxygen was supplied to the cells through thin walled silicone tubing. The mouse-mouse hybridoma cells were grown in three identical bioreactors at perfusion rates of 1.1, 2.0, and 3.2/day for a period of eight days during which the viable cell concentrations reached stable values of 2.6×10⁶, 3.5×10⁶, and 5.2×10⁶ cells/ml, respectively. Total cell densities reached values ranging from 8×10⁶ to 1×10⁶ cells/ml. Specific substrate consumption and product formation rates responded differently to changes in cell density and apparent specific growth rate, which were not varied independently. Using multiple regression analysis, the specific glucose consumption rate was found to vary with viable cell density while the specific glutamine uptake and lactate production rates varied with both viable cell density and apparent specific growth rate. These results suggest that cell density dictates the rate of glucose consumption while the cell growth rate influences how glucose is metabolized, i.e., through glycolysis or the TCA cycle. The specific antibody production rate was found to be a strong function of cell density, increasing as cell density increased, but was essentially independent of the specific growth rate for the cell line under study.List of Symbols MAb monoclonal antibody - X _v viable cell density (cells/ml) - X _d nonviable cell density (cells/ml) - specific growth rate (1/day) - k _d specific death rate (1/day) - D dilution rate (1/day) - S _f substrate concentration in feed (g/l or mM) - S substrate concentration (g/l or mM) - P _f product concentration in feed (g/l or g/ml) - P product concentration (g/l or ug/ml) - q _s specific consumption rate of substrate (g/hr/cell or mmol/hr/cell) - q _p specific production rate of product (g/hr/cell) - q _MAb specific production rate of monoclonal antibody (g/hr/cell) This work was supported in part by a grant for the National Science Foundation (BCS-9157851) and by matching funds from Merck and Monsanto. We sincerely thank Mr. Roland Buchele of Akzo Inc. (Germany) for donation of the polypropylene membranes, Dr. Michael Fanger (Dartmouth Medical School) for the hybridoma cell line, Dr. Sadettin Ozturk (Verax Corp., Lebanon, NH) for technical discussions regarding reactor design, and Dr. Derrick Rollins (Iowa State University) for advice on statistical methods.     

A light microscopic study of nuclear and cytoplasmic size of the aggregate acidophil population in the hypophysis cerebri,pars distalis,of adult male mule deer,Odocoileus hemionus hemionus,relative to seasons of the photoperiod and antler cycles

Summary Nuclear and cytoplasmic size (²) of acidophils in the pars distalis of adult male deer were measured and the data analyzed statistically with reference to the four classical seasons of the photoperiodic cycle and five periods and two events of the antler cycle. The seasons, and the respective mean cross-sectional areas of cell nuclei and cytoplasm, are: Spring (21 March–20 June; increasing photoperiod greater than 12 hours), 28.45 and 48.68 ²; Summer (21 June–20 September; decreasing photoperiod greater than 12 hours), 27.81 and 44.25 ²; Fall (21 September–20 December; decreasing photoperiod less than 12 hours), 30.40 and 45.07 ²; Winter (21 December–20 March; increasing photoperiod less than 12 hours) 27.28 and 40.30 ². Beginning in late winter and early spring the components of the antler cycle, and the respective cross-sectional areas of cell nuclei and cytoplasm, are: Initial Antler Growth, 28.08 and 38.58 ²; Growth of Velvet Antler Form, 28.93 and 50.61 ², Antlers Hardening, 27.53 and 40.83 ²; Velvet Shedding, 29.03 and 53.20 ²; Rutting Season, 29.95 and 44.97 ²; Preparation for Shedding, 26.50 and 40.38 ²; Antlers Recently Shed, 27.53 and 47.85 ². Shedding of boney antlers, growth of velvet antlers and increases in nuclear and cytoplasmic areas of acidophil cells in the pars distalis occur when the photoperiod is increasing. Construction and retention of hard antlers occur when photoperiod is decreasing. This lessening of day length appears to influence the gonadotrophs that regulate secretion of testosterone.A part of a dissertation submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy, Anatomy, Colorado State University. Specimens were contributed by Colorado Federal Aid Project W-105-R, Colorado Division of Game, Fish and Parks, Game Research Center, Fort Collins and investigations of them was supported by grant T1-DE130 from the National Institutes of Health, Bethesda, Maryland, U.S.A.NIDR Predoctoral Trainee.     

Cyclin-dependent kinase inhibitor,roscovitine, in combination with exogenous cytokinin,N6-benzyladenine,causes increase of cis-cytokinins in immobilized tobacco cells

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N ⁶-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N ⁶-Benzyladenine (1.25 M) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 M) further increased cis-cytokinins, up to 2.2 nM.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.