Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Determination of the effective diffusion coefficient of oxygen in gel materials in relation to gel concentration

Summary The effective diffusion coefficient of oxygen, ID_e, was determined in different gel support materials (calcium alginate, -carrageenan, gellan gum, agar and agarose) which are generally used for immobilization of cells. The method used was based upon fitting Crank's model on the experimental data. The model describes the solute diffusion from a well-stirred solution into gel beads which are initially free of solute. The effect of the gel concentration on ID_e of oxygen in the gel was investigated. The results showed a decreasing ID_e for both agar and agarose at increasing gel concentration. In case of calcium alginate and gellan gum, a maximum in ID_e at the intermediate gel concentration was observed. It is hypothesized that this phenomenon is due to a changing gelpore structure at increasing gel concentrations. The ID_e of oxygen in calcium alginate, -carrageenan and gellan gum varied from 1.5^*10^–9 to 2.1^*10^–9 m²s^–1 in the gel concentration range of 0.5 to 5% (w/v).     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Effect of gelation temperature and gel-dissolving solution on cell viability and recovery of two Pseudomonas putida strains co-immobilized within calcium alginate or κ-carrageenan gel beads

Summary Since a lethal effect of an increased temperature (42°C) on Pseudomonas putida strains PaW8 and PaW130 was demonstrated, strictly ionotropic gels such as calcium alginate or -carrageenan type X 0909 were used for cell co-immobilization, rather than a thermoionotropic -carrageenan gel. Among the variety of gel-dissolving solutions tested, only a 0.05M Na₂CO₃/0.02M citric acid solution was able to preserve around 100 % of the cell viability. A complete cell recovery was obtained from calcium alginate gel beads, while only 6 % of viable cells was recovered from the ionotropic -carrageenan gel.     

Influence of growth behaviour and physiology of alginate-entrapped microorganisms on the oxygen consumption

Summary The ability of alginate-entrapped microorganisms to supply oxygen was determined with regard to physiology and growth behavior of the cells. Oxygen diffusion through an alginate film containing different concentrations of Pseudomonas putida or Saccharomyces cerevisiae was measured. Oxygen diffusion decreased when the cell loading increased. Dependent on the physiological behavior of these organisms the course of the oxygen concentration under the gel film is quite different. In further experiments an Effectiveness-Factor of oxygen uptake of alginate beads with Saccharomyces cerevisiae or Aspergillus niger was determined in relation to the growth behavior of the organisms. The effectiveness factor is always higher when the biomass is concentrated in the outer region of the gel beads as if the microorganisms are distributed homogeneously in the alginate. Considering these results it is not possible to make a general statement on the ability of microorganisms in alginate to supply oxygen. The physiology and the growth behavior of the immobilized organisms have to be considered in any case.     

Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate

Different factors which affect the stability of calcium alginate gel beads entrapping viable cells during fermentation were investigated. It was found that among others, the initial population of cells per ml of gel beads, the length of period of incubation in CaCl₂ solution, and the concentration of sodium alginate used for the immobilization were the most important factors affecting the stability of the gel beads during fermentation. By using an initial cell population of about 10⁵ cells per ml of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCl₂ solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCl₂ into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.     

Culture of chondrocytes in alginate gel: Variations in conditions of Gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes

Summary Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.     

Stable ionotropic gel for cell immobilization using high molecular weight pectic acid

It is shown that Ca- and Al-pectate gel beads prepared by use of high molecular weight polygalacturonic acid (viz. polygalacturonic acid having a high STAUDINGER index) are well suited for cell immobilization. The pectate beads are much more insensitive to those ions and chemical agents which destructively act on alginate beads (such as phosphate, citrate, gluconate, lactate as well as a high excess of sodium, potassium, and/or ammonium ions), even without addition of gelling cations to the liquid phase.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

Phosphorus foraging root development of Brassica rapa nothovar. grown in a phosphorus-deficient nonallophanic Andisol

Lateral root of Brassica crops firmly aggregated around Ca-alginate gel beads containing dicalcium phosphate dihydrate and -cyclodextrin (DCPD gel bead) in a phosphate (P)-deficient soil (Nanzyo et al., 2002, Soil Sci. Plant Nutr. 48, 847–853). The first aim of the present study was to identify the component in the DCPD gel beads that accounts for the special root proliferation. This P-foraging root growth was observed in plots applied with either polyolefin-coated NH₄H₂PO₄ (POC-MAP) or DCPD powder instead of the DCPD gel beads. The POC-MAP neither contains Ca, alginate nor -cyclodextrin. The DCPD powder was applied in a similar number of spots with the number of DCPD gel beads. Thus, the essential component in the DCPD gel beads for the P-foraging root growth around them was P. The second aim was to examine the effect of various inorganic P sources on the P uptake of B. rapa nothovar. While significant P uptake was obtained in the plot applied with apatite from Florida, USA, sediment origin (F-Ap), almost no P uptake was obtained in that with apatite from Quebec, Canada, igneous origin in the P-deficient nonallophanic Andisol. Hence, a P-release level from F-Ap was near the lower limit for the P uptake by the B. rapa nothovar. under the present experimental conditions. These results indicate the P foraging characteristics of the Brassica roots contribute to improve the P recovery rate in the agricultural fields with localized application of moderately-soluble P fertilizers.     

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, -carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.     

Conductimetric assessment of the biomass content in suspensions of immobilised (gel-entrapped) microorganisms

Summary The problem of obtaining a rapid estimate of the microbial content of an immobilised cell suspension is addressed. The low-frequency conductivity of free-living cell suspensions of Clostridium pasteurianum is lower than that of the medium in which they are suspended, by an amount conforming to the Bruggeman relation. The conductivity of the cell wall makes a negligible contribution to the measured conductivity under the conditions used. Calcium alginate beads (lacking microbial cells) lower the conductivity of a solution with which they have been equilibrated by an extent which is a function of the concentration of alginate gel used in forming the beads. When this is taken into account, the ratio of the conductivity of a suspension of gel-immobilised cells to that of the suspending medium can be used to give a rapid and convenient assessment of the amount of microbial biomass present.     

Preparation of stable alginate gel beads in electrolyte solutions using Ba2+ and Sr2+

Summary Alginate solutions with two different M/G (mannuronic acid/guluronic acid) ratios were added dropwise to SrCl₂ and BaCl₂ solutions. The low M/G ratios (0.27) Sr^– and Ba-alginate gel beads were more chemically and physically stable in electrolyte solutions than conventional Ca^– alginate gel beads. These gel beads with immobilized yeast cells had normal ethanol productivities.author to whom all correspondence should be addressed.     

Interactions between a genetically markedPseudomonas fluorescens strain and bacteriophage ΦR2f in soil: Effects of nutrients, alginate encapsulation, and the wheat rhizosphere

The introduction of bacteriophages could potentially be used as a control method to limit the population size of engineered bacteria that have been introduced into soil. Hence, the ability of a species-specific phage, R2f, to infect and lyse its host, a Pseudomonas fluorescens R2f transposon Tn5 derivative, in soil, was studied. Control experiments in liquid media revealed that productive lysis of host cells by phage R2f occurred when cells were freely suspended, whereas cells present in alginate beads resisted lysis. The presence of nutrients enhanced the degree of lysis as well as the production of phage progeny, both with the suspended cells and with cells escaped from the alginate beads. Experiments in which host cells and phage R2f were introduced into two soils of different texture revealed that host cells were primarily lysed in the presence of added nutrients, and phage reached highest titres in these nutrient-amended soils. Encapsulation of the host cells in alginate beads inhibited lysis by the phage in soil. Populations of free host cells introduced into soil that colonized the rhizosphere of wheat were not substantially lysed by phage R2f. However, P. fluorescens R2f populations colonizing the rhizosphere after introduction in alginate beads were reduced in size by a factor of 1,000. Cells migrating from the alginate beads towards the roots may have been in a state of enhanced metabolic activity, allowing for phage R2f infection and cell lysis. Correspondence to: J.D. van Elsas     

Effect of glycerol and PEGMA coating on the efficiency of cell holding in alginate immobilized <Emphasis Type="Italic">Synechococcus elongatus</Emphasis>

Synechococcus elongatus cells were immobilized in alginate beads, and the effects of increasing the cross-linker concentration from 2 to 4 % CaCl₂ were evaluated, as well as the effects of coating the beads with either glycerol or poly(ethylene glycol) methyl ether methacrylate (PEGMA)—not previously reported for immobilized microalgae—to improve the holding time of the immobilized cells. S. elongatus cells remain metabolically active after coating with glycerol or PEGMA. There is an inverse relation between the glycerol concentration and the chlorophyll a content for the alginate beads cross-linked with 2 % CaCl₂. PEGMA diminishes the rate of liberation of cells as its concentration increases, although results suggest the ability of S. elongatus to degrade PEGMA, which increases the growth rate of the liberated cells, because of PEGMA being used as carbon source.     

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

Summary The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl₂ within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.     

Investigation of alginate gel inhomogeneity in simulated gastro-intestinal conditions using magnetic resonance imaging and transmission electron microscopy

The inhomogeneity of alginate gel beads prepared by an external diffusion method has been characterised using spatially resolved nuclear magnetic resonance or “magnetic resonance imaging” (MRI) and transmission electron microscopy (TEM). The beads exhibited various degrees of inhomogeneity although reducing the length of exposure to the gelling bath and the presence of non-gelling ions decreased gel inhomogeneity. In order to gain information regarding the gastro-intestinal functionality of these beads for in vivo applications, they were exposed to simulated gastro-intestinal conditions. The increased polymer concentration at the edge of the beads was shown to persist throughout our gastro-intestinal model despite the centre of the bead becoming progressively more porous in nature. The porosity of the alginate gels has been quantified by image analysis of transmission electron micrographs and shown to depend on both location within the bead and gastro-intestinal conditions. We suggest that such changes in porosity of these alginate beads during simulated gastro-intestinal conditions may make these an attractive option for controlled delivery applications in vivo.     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Prevention of limiting substrate diffusion in an immobilized enzyme reaction system: Lectin-induced activation of gel-entrapped β-D-galactosidase

Summary Escherichia coli -D-galactosidase (EC 3.2.1.23) was entrapped in polyion complex-stabilized alginate gel beads together with a lectin fromRicinus communis (RCA₁ lectin). The rate of entrapped enzyme-catalyzed hydrolysis of O-nitrophenyl--D-galactoside dramatically increased with an increase in lectin content, and at the maximum level of lection content the entrapped enzyme activity exceeded the native enzyme activity. A rapid decrease in the apparent K_m was observed while increasing the lectin content, whereas the V_max value varied insignificantly.