Physical Studies on Cell Immobilization Using Calcium Alginate Gels

Columns of calcium alginate gel pellets have excellent physical properties when used as a cell immobilization support. Columns of pellets were very resistant to compression and abrasion during passage of high concentrations of sucrose at high flow rates, but if the pellets were formed using low alginate and Ca²⁺ concentrations, compression occurred and flow out of the column was reduced and pressure built up. Transfer of sucrose into the pellets was controlled by internal diffusion, the rate of diffusion being increased by reductions in the alginate and Ca²⁺ concentrations used for immobilization and by the presence of entrapped active cells. Some leakage of cells occurred during use especially when cell division of the entrapped cells took place, but leakage could be minimized by using more highly polymerized pellets. Therefore, immobilization conditions can be chosen so as to form strong pellets, possessing high substrate transfer rates and low rates of cell leakage.     

Alumina-doped alginate gel as a cell carrier for ethanol production in a packed-bed bioreactor

Alumina-doped alginate gel (AEC) was developed as a new type of cell carrier to be used in ethanol fermentation. The presence of the alumina particles in alginate gel not only improved the porous structure of the carrier, but also provided many advantageous characteristics including good mechanical strength, high stability, and high immobilization yield. The attachment of alumina particles and yeast cells by electrostatic attraction was shown to promote cell growth and increase ethanol productivity. The AEC carrier was found to be more effective for the immobilization of Saccharomyces cerevisiae M30 than the conventional Ca-alginate bead. Ethanol productivities of 1.4 and 7.9 ∼ 12.6 g/(L/h) were obtained using the AEC cultures in batch and continuous modes of operation, respectively, with an ethanol yield of 43.9 ∼ 46.7% and an immobilized yield of 81.4 ∼ 84.5%. Ethanol fermentation in a continuous packed-bed reactor using the AEC carrier was stable for > 30 days.     

Immobilization of animal cells using photo-crosslinkable resin

A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody, although other photo-crosslinkable resins had significant inhibitory effects. Using BIX12-alginate hybrid gel particles, hybridoma cells could grow in the resins and produce monoclonal antibody. For the continuous production of monoclonal antibody, perfusion culture using a fluidized-bed bioreactor with direct air bubbling was carried out. By this cultivation, monoclonal antibody could be produced stably for more than 50 d. A high viable cell density of more than 10⁷ cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask. Anchorage-dependent cells were also immobilized in the resin particles by three immobilization procedures. Among these procedures, porous BIX12 formed by adding gelatin powder provided good support strength and allowed the cells to grow on the surface inside of the support.     

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

Summary The conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.     

Growth of an adherent continuous cell line entrapped in a peg/alginate matrix

Summary CHO-K1 cells, an anchorage-dependent line, were entrapped in beads prepared from a Na alginate/polyethylene glycol mixture and grown, through successive passages, to an average maximum density of 4.5×10⁷ viable cells/g of bead. Cell growth and viability was unaffected by repeated alginate re-solubilization and reformation of the gel beads through five passages.     

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.     

Immobilization of animal cells in fixed bed bioreactors

Immobilization of animal cells has become a highly popular means of achieving high-density animal cell cultures. The advantages of immobilization are that it stabilizes cells in culture and enables long-term culture periods to be achieved. Immobilization increases cell productivity by increasing the usable substrate surface area for anchorage-dependent cells, or by facilitating perfusion of anchorage-independent cells. A method for production of secreted biological products from anchorage-dependent and independent cells is described. The method is based on immobilization of animal cells within the polymeric matrix of polyurethane foam, packed in a fixed bed bioreactor.     

Oxygen uptake rate of immobilized growing hybridoma cells

Summary The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.     

Immobilized salt-tolerant yeasts: application of a new polyethylene-oxide support in a continuous stirred-tank reactor for flavour production

Immobilization of salt-tolerant yeasts considerably decreases the total time required for the flavour development in soy-sauce processes. For immobilization of cells, alginate gel is mostly used as support material. However, alginate is not very suitable for use in soy-sauce processes because alginate is sensitive to abrasion and chemically unstable towards the high salt content of the soy-sauce medium. In contrast, a newly developed polyethylene-oxide gel seems to be more suitable, but this gel has not been used so far for flavour production in a bioreactor with a high salt content. Therefore, this gel was applied with immobilized salt-tolerant yeasts in a continuous stirred-tank reactor, containing more than 12.5% (w/v) salt. In this reactor, the polyethylene-oxide gel particles did not show any abrasion for several days, while alginate gel beads were already destroyed within 1 day. In addition, the polyethylene-oxide gel particles with immobilized salt-tolerant yeasts Candida versatilis and Zygosaccharomyces rouxii showed a good flavour production. From this work, it was concluded that the application of polyethylene-oxide gel in long-term soy-sauce processes is attractive in the case the sticking together of polyethylene-oxide gel particles can be controlled.     

Immobilized soy-sauce yeasts: development and characterization of a new polyethylene-oxide support

Entrapment of cells in alginate gel is a widely used mild immobilization procedure. However, alginate gel is not very suitable for use in long-term continuous soy-sauce processes because alginate is sensitive to abrasion and chemically unstable towards the high salt content of soy-sauce medium. Therefore, a chemically crosslinked polyethylene-oxide gel was used instead. The disadvantage of this gel was that due to the crosslinking reaction, the viability of the cells after immobilization was poor. For this reason, a new mild procedure for immobilizing soy-sauce yeasts in polyethylene-oxide gel was developed, resulting in high survival percentages of the soy-sauce yeasts Zygosaccharomyces rouxii and Candida versatilis. This newly developed polyethylene-oxide gel, unlike alginate gel, appeared not to be sensitive to abrasion, even in the presence of high salt concentrations. Therefore, we concluded that this newly developed polyethylene-oxide gel is more suitable than alginate gel for use as immobilization material in long-term processes with a high salt content, like soy-sauce processes.     

Mercury removal by immobilized algae in batch culture systems

The accumulation and volatilization of mercury by non-immobilized and immobilizedChlorella emersonii have been studied in batch culture systems. Reduction in the mercury concentration in the growth medium by non-immobilized cells was highly dependent on inoculum density, whilst reduction in mercury concentration by immobilized cells was rapid at all inoculum densities. Mercury accumulation by immobilized cell biomass was significantly greater than by non-immobilized cells with 10⁶ and 10⁵ cells bead^–1 or ml^–1. Volatilization of mercury by non-immobilized cell systems was greatest at higher inoculum densities, whereas more mercury was volatilized from immobilized cell systems at lower inoculum densities, and was greatest with unstocked alginate beads. Thus, in immobilized systems, mercury removal from solution is complex and involves mercury accumulation by the cells and volatilization by the matrix and cells. Further studies of mercury accumulation and volatilization by unstocked immobilization matrices revealed that agarose volatilized much less mercury than alginate or agar. The precise mechanism of mercury volatilization by alginate remains unclear, though it is thought to be a chemical effect.     

Living immobilized Acetobacter in Ca-alginate in vinegar production: Perliminary study on optimum conditions for immobilization

Summary The optimum conditions forAcetobacter immobilization were investigated. The results show that: 1) the maximum oxygen uptake rate (OURm) and cell release are related to alginate and cell concentration in the gel; 2)different alginate concentration does not affect cell viability, but long storage in CaCl₂ reduces the number of living cells; 3)the double alginate gel layers had no influence on cell viability and on the OURm and prevented cell leakage from the gel matrix.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

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.     

Gel particles from spray-dried disordered polysaccharides

The formation of gel particles from alginate and ι-carrageenan was studied through a novel pathway of formation via an amorphous spray-dried intermediate. Dried biopolymer particles were suspended in solutions of different Ca²⁺ concentration. Particle size ranges and microscopic observation demonstrated that a range of swelling behaviour could be induced, with lower calcium concentrations resulting in more expanded particles, until a lower limit is reached below which particles initially dissolve. For the same calcium charge stoichiometry, larger swollen gel particles were obtained for alginate than for ι-carrageenan. The ability to produce a range of swollen biopolymer gel particle sizes, on the order of 1–600 μm, is attributed to the balance between gelation and dissolution kinetics, with fast gelation kinetics and slow dissolution promoting production of small gel particles whilst fast dissolution with slow gelation leads to larger gel particles. By controlling the solution environment in which rehydration is carried out, it is therefore possible to produce particles with desired degrees of swelling from a single starting material.     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Immobilization of hybridoma cells with alginate and urethane polymer and improved monoclonal antibody production

Summary Hybridoma cells producing anti--amylase monoclonal antibody were entrapped in calcium alginate and the gels were then coated with urethane polymer. The urethane coating improved gel strength and prevented cell leakage. This immobilization method enabled direct air bubbling in the serum-free medium and a very high cell concentration (3×10⁷ cells/ml gel) was obtained. By using a fluidized-bed reactor, effective removal of the medium in addition to sufficient oxygen supply could be achieved without any special devices and a very high concentration of the monoclonal antibody was continuously obtained.     

A novel method for immobilization of yeast cells in alginate gels of various shapes by internal liberation of Ca-ions

Summary In situ gelation of alginate, in which Ca-ions are liberated internally in the gel, was used for immobilization of yeast cells. Compared to the traditional alginate gelation method, internal gelling gave immobilized yeast particles of higher strength, without reduction in fermentation rate.     

Alginate immobilization of recombinant Escherichia coli whole cells harboring l-arabinose isomerase for l-ribulose production

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis l-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca²⁺ concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L⁻¹, respectively. The interactions between Ca²⁺ concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for l-ribulose production, the productivity was as high as 56.7 g L⁻¹ h⁻¹ at 400 g L⁻¹ substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of l-ribulose, particularly because of its high stability and low cost.