Production of rifamycin B and SV by free and immobilized cells of Amycolatopsis mediterranei

The effect of growth conditions (incubation time, inoculum size, initial pH value) and some nutrient concentrations on the growth and rifamycin B and SV production by free and immobilized cells of Amycolatopsis mediterranei CBS 42 575 was studied. In alginate beads, the immobilized cells behaved like the free cells, but a pronounced difference was observed in antibiotic production and cell growth. The rifamycin production by the immobilized cells was higher than that obtained by the free cells. The immobilized cells were also reused repeatedly for six batch cultivations with a fresh medium charged into flasks at the beginning of each batch. It was found that the immobilized cells were stable, and the rifamycin yield was almost constant during the first three batches and then decreased.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation.

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Immobilized serotonin: a novel substrate for cell culture

Baby hamster kidney cells, bovine aortal endothelial cells, bovine smooth muscle cells, and chick embryo fibroblasts were all observed to attach and grow on serotonin which had been immobilized by covalent coupling to agarose beads. While growth and morphology of cells on immobilized serotonin appeared normal, a change in cell function may have occurred since the pattern of polypeptides expressed by these cells was different from that of cells grown on two other substrates: immobilized fibronectin and tissue culture plastic. By changing the composition of the fetal calf serum proteins in the growth medium it was shown that cells attach directly to immobilized fibronectin without mediation by medium components. In contrast, cells were found not to attach directly to immobilized serotonin but to attach indirectly via factors absorbed onto immobilized serotonin from fetal calf serum. The major component of this cell attachment activity was shown not to be fibronectin and was identified following separation by SDS-PAGE, electroblotting, and cell binding on nitrocellulose filters. The cell attachment activity compromises a major protein species of Mr 70,000 which is the molecular size of the recently identified serum spreading factor also called vitronectin.     

Studies on immobilized Saccharomyces cerevisiae. III. Physiology of growth and metabolism on various supports

In earlier communications general analyses of rapid ethanol fermentation by Saccharomyces cerevisiae immobilized on inert supports were described. In this article physiology of growth and metabolism (parameters like rates of CO(2) evolution and O(2) uptake, respiratory quotient, and generation time) of Saccharomyces cerevisiae immobilized on different supports are reported. Values of the ratio of specific oxygen uptake rate for immobilized cells to free cells have been found to be 0.732, 0.781 and 0.785 for carrier A, carrier B, and covalently crosslinked controlled pore glass (CPG, specific surface area of 439 m(2) g(-1)), respectively. Rates of specific CO(2) evolution for immobilized cells to free cells for these supports are 0.784, 0.822, and 0.783, respectively. Marked reduction in generation time of Saccharomyces cerevisiae on all the supports has been observed. No change in size (4.8-5 mum) and specific growth rate (mu(m) = 0.275 h(-1)) of cells leaving the reactor has been observed.     

Immobilization of neural cells in three-dimensional matrices for biosensor applications

To overcome logistical difficulties with current designs of cell- or tissue-based biosensors which have individual cells or tissue slices immobilized on membranes or microelectrode arrays, we have proposed a system that uses three-dimensional cultures of neural cells immobilized in hydrogel matrices. In this design, immobilized cells would be maintained in a reservoir and then transferred to a detector platform when needed for analysis. The development of such a system relies upon a renewable supply of cells and the ability to culture cells for long periods of time in three-dimensions while maintaining their physiological function. To investigate the ability to culture neural cells in 3D matrices, embryonic rat cortical neurons and astrocytes were immobilized by matrix entrapment in a novel sugar poly(acrylate) hydrogel and collagen gels. The sugar poly(acrylate) hydrogel does not appear to support neural cell growth as a result of a lack of cell adherence, small pore size and, possibly, harshness of synthesis conditions. In contrast, collagen gels support the growth of cortical neurons, astrocytes, as well as neural progenitor cells. Evidence is also presented from immunocytochemistry and patch-clamp measurements which shows that neural progenitor cells proliferate in culture and can be induced to differentiate into neural cell types. Thus, they potentially represent a renewable cell source.     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

The Effects of Immobilization on Photosynthesis,Growth, Nitrogen Fixation and Ammonium Ion Excretion of Mutant and Wild Type Cells of Anabaena variabilis

Ammonium ion photoproduced by nitrogen-fixing cyanobacteria is formed from water, air nitrogen and solar energy under normal temperature and atmospheric pressure. It is reported here that the mutant and its parent wiid type cells of Anabaena variabilis from Shanmugan lab were immobilized into the polyurethane foams. The growth curves measured by cell counting, O.D750 measurement and ch]orophyll a determination showed that the growth of the immobilized wild type cells was more rapid than the immobilized mutant cells, and for both two types of cells the free-living cells grew better than the immobilized ones. The nitrogenase activity (reduction of C2H2) was 44% greater in immobilized mutant cells and 232% greater in immobilized wild type cells than both free-living cells, respectively. Free-living cells of wild type A. variabilis almost did not excrete any ammonium ion and the immobilized cells of the wild type had the ammonium ion excretion activity similar to that of the free-living mutant cells. Moreover, immobilization stimulated the activity of ammonium ion excretion 55.6% greater than the free-living cells, in the mutant strain. The fluorescence enhancement induced by DCMU (3-(3,4-dichlor-benzene)-1, 1-dimethl urea) indicated the photosynthetic capability of wild type ceils in both free-living and immobilized states were higher than that of the mutant cells, and the free-living cells of both two types of cells had higher capability than the immobilized cells. The fluorescence emission spectra at 77K showed that there were four peak emission: F646(F645 or F650) and F662(F664 or F665) from phycobilin, F693(F698,F700 or F702) from PS Ⅱ and F732(F728 or F733 from PS Ⅰ In both two types of immobilized cells the photosynthetic light energy distribution tended to be in PS Ⅰ and it seems to be benefitlal for the nitrogenase activity and ammonium ion excretion. SEM observation indicated that the shape and size of the immobilized cells remained unchanged incompared with the free-living cells, However, the surface of the immobilized cells had accumulated some mucilage, and particularly, the film of mucilage coated both surface of the mutant cells and the foam matrix.     

Oxygen uptake by entrapped hybridoma cells

Oxygen consumption by hybridoma cells immobilized in 1- and 3.9-mm-diameter calcium alginate beads was measured. The entrapped cells consumed oxygen at about 10 mumol/min per 10(9) cells, regardless of the bead size and cell loading. In contrast, the same cells in suspension culture respire at specific rates of 3-8 mumol/min per 10(9) cells (depending on the cell density). The growth rate of the immobilized cells was significantly reduced, while specific antibody production was comparable to that of free cells.     

Growth and removal of nitrogen and phosphorus by free-living and chitosan-immobilized cells of the marine cyanobacterium Synechococcus elongatus

This study investigated the growth rate of chitosan-immobilized cells of the marine cyanobacterium Synechococcus elongatus and its potential application in the removal of nitrogen and phosphorus for wastewater treatment. Immobilized cell cultures had a lag phase of growth due to the immobilization method, and their growth rate was similar to that of free-living cell cultures. Ammonia removal was higher in free cells (54%) than in immobilized cells (29%), but nitrate removal was similar in immobilized (38%) and free cells (44%); phosphorus removal was more efficient in free cells (88%) than in immobilized cells (77%). Chlorophyll a and protein content were higher in immobilized cells. Our study demonstrates that S. elongatus immobilized into chitosan capsules can remove nutrients and is able to maintain a growth rate comparable to that of free cells in culture.     

Autoradiographic determination of mass-transfer limitations in immobilized cell reactors

Pseudomonas putida cells were grown in confined volumes in dual-membrane immobilized cell reactors constructed from microporous polyethylene hollow fibers and silicone rubber tubules as a model system for the study of mass transport in microbial aggregates. Local cell concentrations in the reactors reached 300 g dry mass/L. Pulse-chase radioisotope labeling with (35)SO(4) (2-) was used to estimate the rates of cell mass synthesis and degradation. Sulfur incorporation consistently exceeded sulfur release, implying that the cell mass concentration continually increases. The location and size of the cell growth region was determined using liquid emulsion autoradiography of thin sections prepared from labeled reactors. Cell growth occurs in a region less than 25 mum in depth adjacent to the oxygen supply, and the expansion of the cells caused by cell growth promotes convection of the cell mass into regions of the reactor where starving cells accumulate. The combination of mass-balance and spatial distribution measurements that can be made using radioisotope tracers provides a versatile method for determining metabolic rates and limitations caused by mass transfer in immobilized cell reactors.     

Clues to the origin of high external invertase activity in immobilized growing yeast: prolonged SUC2 transcription and less susceptibility of the enzyme to endogenous proteolysis.

Expression of the SUC2 gene encoding invertase was studied using free and gelatin-immobilized yeast cells to try to explain the high activity of this enzyme exhibited by immobilized cells when allowed to grow in a nutrient medium. The results indicated that at least two factors are probably responsible for the accumulation of invertase in immobilized cells. First, the expression of the SUC2 gene was maintained throughout growth in immobilized cells, whereas its expression was only transient in free cells. Second, invertase of immobilized cells was shown to be less susceptible to endogenous proteolytic attack than that of the corresponding free cells. These results have been interpreted, respectively, in terms of diffusional limitations and changes in the pattern of invertase glycosylation due to growth of yeast in an immobilized state.     

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.     

Cell mass synthesis and degradation by immobilized Escherichia coli

Escherichia coli K-12 cells were grown in a confined volume using microporous hollow fiber membranes. The local cell concentrations in the reactors were above 400 g dry mass/L, in excess of the predicted limit based on the specific volume of free cells determined by tracer exclusion. Cell mass synthesis and degradation rates in these reactors were measured using radioisotope labeling with (35)S. Net accumulation of cell material persisted at these high cell densities. The rates of substrate uptake and cell growth were predicted from the theory of reaction and diffusion assuming that kinetics of cell metabolism are identical for free-living and immobilized cells. This theory was tested by comparison of overall rates and by the size of the region in which cell growth occurred, measured by autoradiography. A yield coefficient of 4 +/- 1 mol sulfur/mol glucose was measured, in agreement with the value determined for free-living cells in similar conditions. Cell growth occurs in a thin layer (10-30 mum), at a rate similar to the growth rate for free cells. Volume expansion by the cells as a consequence of proliferation induces convection of cell mass out of the growth region into a region of the reactor filled with starving cells, which then accumulate in the reactor. The combination of mass-balance and spatial distribution measurements made possible by the use of radioisotope labeling enables a direct test for mass transfer limitations, the determination of the intrinsic cell kinetics, and noninvasive measurements of cell growth in immobilized cell reactors.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

Production of minimally disturbed synchronous cultures of hematopoietic cells

A method is describedforproducing sizable quantities of synchronously dividing, minimally disturbed mammalian cells. Cultures were grown immobilized on surfaces such that cell division within the population resulted in the continuous release of synchronous newborn cells. As judged by the quality and duration of synchronous growth, cell size distributions, and DNA compositions, newborn mouse L1210 cells grew with a very high level of synchrony without overt evidence of growth disturbances. The technology should be applicable to a variety of hematopoietic cells, as evidenced by similar results with human MOLT-4 and U937 cell lines.     

Growth kinetics of microalgae in microfluidic static droplet arrays

We investigated growth kinetics of microalgae, Chlorella vulgaris, in immobilized arrays of nanoliter‐scale microfluidic drops. These static drop arrays enabled simultaneous monitoring of growth of single as well as multiple cells encapsulated in individual droplets. To monitor the growth, individual drop volumes were kept nearly intact for more than a month by controlling the permeation of water in and out of the microfluidic device. The kinetic growth parameters were quantified by counting the increase in the number of cells in each drop over time. In addition to determining the kinetic parameters, the cell‐size distribution of the microalgae was correlated with different stages of the growth. The single‐cell growth kinetics of C. vulgaris showed significant heterogeneity. The specific growth rate ranged from 0.55 to 1.52 day^?1 for different single cells grown in the same microfluidic device. In comparison, the specific growth rate in bulk‐scale experiment was 1.12 day^?1. It was found that the average cell size changes significantly at different stages of the cell growth. The mean cell‐size increased from 5.99 ± 1.08 to 7.33 ± 1.3 µm from exponential to stationary growth phase. In particular, when multiple cells are grown in individual drops, we find that in the stationary growth phase, the cell size increases with the age of cell suggesting enhanced accumulation of fatty acids in older cells. Biotechnol. Bioeng. 2012; 109: 2987–2996. © 2012 Wiley Periodicals, Inc.     

Precursor frequency of human T4 cells responding to stimulation through the CD3 molecular complex: role of various cytokines in promoting growth and IL2 production

The frequency of human T4 cells induced to grow and produce IL2 in response to the anti-CD3 mAb, 64.1, was examined. T4 cells were cultured at limiting dilution and stimulated with either soluble or immobilized 64.1 in the presence of various cytokines and/or irradiated B lymphoblastoid cells as accessory cells (AC). The frequency of responding cells was assessed by examining wells microscopically for visible growth and supernatants for IL2. Immobilized, but not soluble, 64.1 was able to induce T4 cells to grow in the complete absence of AC, but only when exogenous cytokines were present. IL2 was most effective at supporting T4 cell growth in this system, with a mean of 26.0 +/- 3.8% of immobilized 64.1-activated T4 cells generating a colony in cultures supplemented with IL2. IL4 could also support the growth of immobilized 64.1-activated T4 cells, but the frequency of responding cells was much lower (3.7 +/- 0.9%). The combination of IL2 and IL4 was not more effective than IL2 alone. TNF alpha, IL1 beta, and IL6 were unable to support T4 cell growth alone, but each increased the frequency of T4 cells responding in the presence of IL2. AC could support the growth of a small number of 64.1-stimulated T4 cells in the absence of exogenous IL2 and enhanced the frequency of T4 cells responding to immobilized 64.1 in the presence of IL2. The percentage of immobilized 64.1-stimulated T4 cells producing IL2 was also examined. Immobilized 64.1 stimulated less than 1.4 in 1000 T4 cells to produce IL2 in the absence of AC and neither IL4 nor TNF alpha enhanced this response. Fixed AC and IL1 beta, on the other hand, caused a small increase in the frequency of immobilized 64.1-activated T4 cells that secreted IL2. The frequency of T4 cells stimulated to produce IL2 by immobilized 64.1 was greatly enhanced by the addition of AC. The data indicate that in the absence of AC, a stimulatory matrix of immobilized 64.1 is sufficient for some T4 cells to be activated to become IL2 or IL4 responsive and for a smaller percentage to secrete IL2. Additional T4 cells require IL1 beta, TNF alpha, IL6, or AC to become IL2 responsive, whereas only IL1 beta and AC can promote IL2 production. In the presence of AC, the amount of cytokine produced endogenously appears to be sufficient to sustain the growth of some T4 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasmid inheritability and biomass production: comparison between free and immobilized cell cultures of Escherichia coli BZ18(pTG201) without selection pressure.

Maintenance of the plasmid pTG201 in Escherichia coli BZ18 was studied for both free and immobilized cells during chemostat culture, in the absence of the antibiotic against which resistance was plasmid encoded. Electron microscopic observations of immobilized proliferant cells within carrageenan gel beads showed high cell concentrations and growth into distinct cavities. The plasmid which coded for the catechol 2,3-dioxygenase activity was stably maintained during 80 generations in the case of immobilized cells. A theoretical analysis founded on the compartmentalization resulting from the immobilized growth conditions was described. However, the model still showed a plasmid stability inferior to that determined experimentally. Hypotheses dealing with physiological changes of immobilized cells were presented. In addition, the high cell concentrations obtained in the outer 50 microns of the carrageenan gel beads gave a biomass productivity within this useful volume which was 20 times higher than in free-cell cultures.