Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

Stability of antibody productivity is improved when hybridoma cells are entrapped in calcium alginate beads

Loss of monoclonal antibody (MAb) productivity in long-term, free-suspended cell culture is often attributed to the appearance of a nonproducing population of hybridoma cell (NP) in the culture which has a growth advantage over the producing population (P). However, when an NP appears in long-term culture of entrapped cells, it may not be able to take over the whole culture in a short period of time due to the limited growth of the entrapped cells. In order to examine the hypothesis that entrapped cells can have improved stability of MAb productivity due to limited cell growth, free-suspended cell culture and calcium alginate-entrapped cell culture with inocula consisting of a P and an NP were compared with regard to stability of MAb productivity in a repeated fed-batch culture. In free-suspended cell culture, the NP appeared to take over the whole culture within three batches, and thereby MAb production completely disappeared. In entrapped cell culture, an NP appeared to outgrow the P rapidly only during an exponential growth phase, resulting in a significant decrease in specific MAb productivity, q(MAb), from 11.58 mug/10(6) cell/day to 2.76 mug/10(6) cell/day. However, when the cell growth was limited in entrapped cell culture, the NP no longer outgrew the P rapidly, as indicated by the stable value of q(MAb). In addition, when the cells recovered from the alginate beads by citrate buffer treatment were subcultured in free-suspended cell culture, MAb production rapidly deteriorated and completely disappeared within two batches. Thus, the P present at a small fraction of viable cell concentration in the beginning of the free-suspended cell culture, which were previously entrapped in alginate beads, seemed to be outgrown rapidly by the NP. Taken together, the results obtained from these experiments support the hypothesis that the limited cell growth in entrapped cell culture, which keeps an NP from taking over the whole culture, is responsible, in part, for the improved stability of MAb productivity. (c) 1993 John Wiley & Sons, Inc.     

Nucleoside triphosphates production using recombinant Escherichia coli entrapped in calcium pectate gel

Nucleoside triphosphates, important intermediates in oligosaccharide synthesis by glycosyltransferases, were generated from nucleoside monophosphates by E. coli BL21(DE3) which over-expresses polyphosphate kinase (PPK) or by Corynebacterium ammoniagenes ATCC 21264 using 1% (w/v) polyphosphate as a phosphate donor and a source of energy. Beads of calcium pectate gel were stable in polyphosphate reaction broth for 60 days after which the activity of PPK was 50% of its original value. This technique could be used for the large-scale synthesis of oligosaccharides.     

Diffusion of sucrose and yohimbine in calcium alginate gel beads with or without entrapped plant cells

The diffusion characteristics of sucrose, a nutrient, and yohimbine, a secondary metabolite, in alginate gel beads, with or without entrapped periwinkle (Catharanthus roseus) or apple (Malus domestica) cells, were investigated. Effective diffusivities of both solutes in the gel beads were determined by two different methods from transient concentration changes in well-stirred solutions where the beads were suspended. The linear plot method developed in this work is easy to use and requires no data from the initial periods of diffusion experiments. It was found that while the cell-free beads provided only minor diffusional resistance to both solutes, the effective diffusivities of both solutes decreased significantly with the presence of cells in the beads and the amount of reduction was proportional to the amount of cell loading. Further, the effective diffusivity of sucrose appeared to be slightly larger than that of yohimbine under identical conditions. It was also observed that permeabilization of apple cells with dimethyl sulfoxide (DMSO) led to an increase in effective diffusivity with the effect being more significant for yohimbine.     

Cream fermentation by a mixed culture of lactococci entrapped in two-layer calcium alginate gel beads

This investigation was directed towards the development of a process which produces a fermented cream of greatly reduced cell number.Lactococcus lactis subsp.Lactis andLactococcus lactis subsp.lactis biovardiacetylactis were entrapped separately in normal or two-layer Ca-alginate gel beads. Pasteurized cream (31% fat content) was inoculated with free-cells and with normal or two-layer beads. When 8% of the total volume was occupied by the gel, there was 300–800 times more inoculum in this system and the fermentation time was considerably reduced (5h against 18h). When pH 5.0 was reached, the residual free-cell count was 150 and 1800 times less than for a classical inoculation method with free-cells for normal and two-layer beads respectively. This result was reproducible for several consecutive runs. Also, the problems linked to storage acidification (souring, wheying-off) appeared later and living lactic acid bacteria were maintained in the product.     

Physical studies on the mechanical stability of columns of calcium alginate gel pellets containing entrapped microbial cells

Constant strain rate and stress relaxation tests on columns of spherical alginate pellets containing entrapped microbial cells demonstrated non-linear viscoelastic behaviour, the columns being relatively resistant to compression over long periods. Compressibility rose with decreases in the alginate concentration used to form the pellets and the soluble sucrose concentration therein, and with increases in temperature and the concentration of cells or other particulate materials; but appeared to be unaffected by the relative dimensions of the column. Pellets were not fractured unless very high pressures were used and deformation was only partially reversible. Over long time periods large creep effects were observed, the rate of compression decreasing exponentially with time. The creep rate increased with the pressure applied, but could be decreased by pumping fluid up the column. Thus the compression of large columns operated continuously for long periods could be modelled by pumping fluid at high flow rates up small columns while applying large pressures to the top of the column. Abbreviation: the ratio of wet weight: dry weight for the yeast cells used in this study is 4:1; weights of cells are always quoted as wet weights.     

Biotransformation of cladribine using a stabilized biocatalyst in calcium alginate beads

Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy-cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled-up reaching a productivity of 138 mg L⁻¹ hr⁻¹. Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions.     

Preparation and properties of glucose isomerase of Streptomyces kanamyceticus cells entrapped in calcium alginate

Summary The properties of glucose isomerase in native, heat-treated and immobilized cells of Streptomyces kanamyceticus after heat and mineral treatment have been compared. The optimum pH for glucose isomerase in native cells was shifted from 8.2 to 8.6 by heat treatment and immobilization. There is no change in the optimum temperature (90°C) for activity of the enzyme by the above treatment. Heat-treated cells and immobilized cells show greater pH and thermal stability of the enzyme. The Km values of the enzyme of native cells, heat-treated cells and immobilized heat-mineral-treated cells are 208 mM, 212 mM and 166 mM respectively; Mg⁺⁺ and Co⁺⁺ enhance the activity of isomerase in all cases.     

A study of cryogenic tissue-engineered liver slices in calcium alginate gel for drug testing

To address issues such as transportation and the time-consuming nature of tissue-engineered liver for use as an effective drug metabolism and toxicity testing model, “ready-to-use” cryogenic tissue-engineered liver needs to be studied. The research developed a cryogenic tissue-engineered liver slice (TELS), which comprised of HepG2 cells and calcium alginate gel. Cell viability and liver-specific functions were examined after different cryopreservation and recovery culture times. Then, cryogenic TELSs were used as a drug-testing model and treated with Gefitinib. Cryogenic TELSs were stored at −80 °C to ensure high cell viability. During recovery in culture, the cells in the cryogenic TELS were evenly distributed, massively proliferated, and then formed spheroid-like aggregates from day 1 to day 13. The liver-specific functions in the cryogenic TELS were closely related to cryopreservation time and cell proliferation. As a reproducible drug-testing model, the cryogenic TELS showed an obvious drug reaction after treatment with the Gefitinib. The present study shows that the cryopreservation techniques can be used in drug-testing models.     

Metabolic characteristics of bacterial cells entrapped in beaded calcium alginate and/or pectate gels

A mixture of heterotrophic bacteria and collection strains ofEscherichia coli andPseudomonas fluorescens were immobilized in calcium alginate or pectate gels. Comparison of respiratory activity, substrate uptake and biosynthetic capacity of immobilized cells showed that both types of carriers permit a prolonged preservation of metabolic activity but the transfer of substances through the gel is faster in the pectate. Morphological changes include some intracellular structures, partial shrinkage of the plasma membrane of immobilized cells, and transformation of a rod-like cell shape to an oval one.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.     

Production of erythropoietin by BHK cells growing on the microcarriers trapped in alginate gel beads

Baby hamster kidney (BHK) cells engineered to produce recombinant human erythropoietin (EPO) were cultured at high density on microcarriers entrapped by calcium alginate gel particles. In this system, the BHK cells proliferated not only on the microcarriers but also in vacant spaces in the alginate gel particles. These spaces contributed greatly to high-density cultivation of the cells and a high productivity of EPO.Abbreviations BHK Baby Hamster Kidney - EPO Erythropoietin     

Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

包埋法固定化真菌漆酶及其应用研究

采用海藻酸钠包埋法固定真菌漆酶,海藻酸钠和CaCl2的最佳浓度分别为3%和4%,最佳给酶量为30U,最大回收率为48.0%.与游离漆酶相比,固定化漆酶的热稳定性有明显改善,最适反应pH向酸性方向漂移0.5,最适反应温度提高了5℃.使用固定化酶处理低浓度造纸废水,运行8批次后残留酶活为64%.     

Appearance of a methylated ribonucleic acid on illumination of Euglena gracillis cells grown in the dark

Investigation of the methylation of nucleic acids by [Me-(3)H]methionine after illumination of Euglena cells grown in the dark has shown that a high-molecular-weight nucleic acid fraction undergoes methylation after exposure to light for 60-120min. This methylated nucleic acid fraction was isolated both by sucrose-density-gradient centrifugation and exclusion chromatography on Sephadex G-200. The fraction was shown to consist of a preformed RNA that is present in cells grown in the dark and which on illumination is transmethylated by methionine.     

Improvement of inulinase stability of calcium alginate immobilized Kluyveromyces marxianus cells by treatment with hardening agents

To improve the inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) stability of calcium alginate-immobilized Kluyveromyces marxianus cells, treatment with hardening agents has been investigated. Treatment of immobilized cells with some polycationic polymers resulted in little decrease in volumetric reactor productivity, but was most effective in increasing the inulinase stability of the immobilized cells. Inulinase stability of glutaraldehyde-hardened immobilized cells increased two-fold, and for hexamethylenediamine + glutaraldehyde and polyethyleneimine + glutaraldehyde-hardened cells increased six-fold compared with that of the unhardened cells.     

Synthesis of functional proteins using Escherichia coli extract entrapped in calcium alginate microbeads

In this report, we describe the construction and analysis of a cell-free protein synthesis system immobilized in calcium alginate microbeads. When incubated in a feeding solution that contained amino acids and other low-molecular-weight substrates, the microbeads transcribed and translated coimmobilized DNA into functional proteins. Protein synthesis continued for more than 15 h with the diffusional supply of substrates and removal of by-products. In addition, functional proteins were generated from PCR-amplified genes as efficiently as from plasmid, suggesting that these cell-like microbeads could be used for functional screening of genomic libraries.     

Coimmobilization of Nitrosomonas europaea and Paracoccus denitrificans cells using polyelectrolyte complex-stabilized calcium alginate gel

Calcium alginate gel (CAG) that withstands phosphate ions in the medium was prepared by reinforcing a network structure of the gel with a polyelectrolyte complex (PEC) consisting of potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide. The PEC-stabilized CAG beads were used as a supporting matrix for the coimmobilization of Nitrosomonas europaea ATCC 25978 cells and Paracoccus denitrificans IFO 12442 cells. The coimmobilized cells were aerobically cultured on a medium containing 3 mM of phosphate ions, using (NH₄)₂SO₄ as a substrate and ethanol as a carbon source. Ammonia was consumed without forming nitrite, indicating the concurrence of nitrification and denitrification in the same system. No breakage of the gel beads was observed during the cultivation. Repeated aerobic cultivation using a column packed with beads of coimmobilized cells had stable initial activity for at least one month.     

Diffusivity of Cu2+ in calcium alginate gel beads

The diffusivity of Cu(2+) in calcium alginate beads calculated by the shrinking core model (SCM) was reevaluated in this work. The results obtained in this work were significantly different than those by the original authors. There were excellent agreements between the results obtained by the SCM in this work and those by the more rigorous linear absorption model (LAM) by the original authors. (c) 1994 John Wiley & Sons, Inc.