Erythromycin production by Streptomyces erythreus entrapped in calcium alginate beads

Summary Mycelia of Streptomyces erythreus were immobilized in calcium alginate beads and employed for production of erythromycin. Compared to conventional and washed mycelial fermentation, the average specific productivity of immobilised mycelia was superior.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Response of field-grownCasuarina equisetifolia to inoculation withFrankia strain ORS 021001 entrapped in alginate beads

A large scale field experiment (ca 1 ha) was carried out in Senegal, to evaluate the response ofCasuarina equisetifolia to inoculation withFrankia strain ORS 021001 entrapped in alginate beads. Biomasses (expressed as dry weight or total nitrogen) of assimilatory branchlets, wood and roots, and nodules were measured in uninoculated and inoculated trees, randomly sampled 1,2 and 3 years after transplantation in the field. When biomasses were expressed as dry weight, increases due to inoculation were similar at the three sampling dates, 45, 36 and 40%, respectively. When biomasses were expressed as total nitrogen, the response to inoculation with time was much higher in the 2nd year than in the 1st and 3rd year. N₂ fixation, estimated using the difference method reached 2.48, 12.25 and 13.44 g N₂ fixed annually per tree. Correspondingly, nodule dry weights, expressed in g per tree, were 2.5, 12.18 and 22.75 at the end of the 1 st, 2nd and 3rd year, respectively. In spite of the positive response of field-grownCasuarina equisetifolia to inoculation, the decrease of N₂ fixation observed in the third year was probably due to unfavorable climatic conditions coupled with insect attacks at the beginning of the third year.     

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.     

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.     

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.     

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.     

Growth of micropropagated bananas colonized by root-organ culture produced arbuscular mycorrhizal fungi entrapped in Ca- alginate beads

The effect of root-organ culture (ROC) produced arbuscular mycorrhizal fungi (AMF), i.e. Glomus proliferum, Glomus versiforme and Glomus intraradices, entrapped in Ca-alginate beads on the first stages development of micropropagated bananas (Musa spp. cv. Grande Naine) was investigated. The experimental design consisted of banana plants inoculated with one of the three AMF and two controls, i.e. Control-AL (with empty alginate beads), and Control (no beads). Forty plants were considered per treatment and cultured under greenhouse conditions in a completely randomized design. Eight plants per treatment were harvested 40, 80, 120, 160 and 200 days after inoculation and analysed for root colonization, growth parameters and nutrient concentration. In addition, spores were enumerated in the substrate at the same intervals. Ca-alginate entrapped ROC-produced AMF spores were able (1) to colonize the root system of a micropropagated banana cultivar under nursery conditions, (2) to increase plant P nutrition and biomass, and (3) to proliferate in the commercial nursery substrate, therefore increasing the fungal inoculum biomass. The entrapment of ROC-propagated spores, adaptable to a wide range of Glomeromycetes, represents thus a forthcoming alternative pathogen-free inoculum.     

Control of an ethanol fermentation carried out with alginate entrapped Saccharomyces cerevisiae

Process control of different reactor models for continuous production of ethanol from sucrose with immobilized yeast has been studied. An enzyme thermistor with immobilized invertase recorded the concentration of sucrose continuously. Ethanol was recorded by a membrane gas sensor with a SnO(2) semiconductor used as detector. A process computer controlled the substrate feed to keep substrate as well as ethanol concentration at preset values by using algorithms of varying complexity. It was thereby demonstrated that PID regulators as well as more advanced algorithms (Otto-Smith regulator, state feedback from a Kalman filter, and cascade control) are useful alternatives to maintain a constant concentration in the fermentor effluents. The time required for the system to return to predetermined conditions after various kinds of disturbances has been especially studied. It was shown that the more advanced regulator used the shorter time.     

Bioconversion of para-substituted monophenolic compounds into corresponding catechols by alginate entrapped cells ofMucuna pruriens

Alginate-entrapped cells ofM. pruriens were able to convert a number of parasubstituted monophenolic compounds into the corresponding catechols. All catechols produced were released into the medium, which offered the opportunity to isolate these products via a relatively simple procedure. Prepurification was performed on a Sephadex G10 gel and catechols were concentrated on Affigel 601. The identity of all products was confirmed with combined liquid chromatography/mass spectrometry (LC/MS) or MS using the desorption chemical ionization technique, depending on the catechol. For the entrapped cells and for a cell homogenate prepared of the same cell line ofM. pruriens the substrate specificities were qualitatively identical when judged on initial rates of synthesis calculated on protein basis.     

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.     

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.     

Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate

Five yeast strains, Saccharomyces cerevisiae D8, M12, and S13; Hanseniaspora uvarum S6; and Issatchenkia orientalis KMBL5774, isolated from Korean grapes, were entrapped in Ca-alginate beads, which are non-toxic, simple to use, and economical. Ca-alginate beads containing yeast cells were soaked in protective solutions, such as skim milk, saccharides, polyols, and nitrogen compounds, before air-blast drying to improve the yeast survival rate and storage ability. The results showed that both entrapment in Ca-alginate beads and soaking in protective agents favorably affected the survival of all strains. The microenvironment formed by the beads and protective agents can protect the yeast cells from harsh environmental conditions, such as low water (below 10 %). All the yeast strains entrapped in Ca-alginate beads showed greater than 80 % survival and less than 11 % water content after air-blast drying at 37 °C for 5 h. In addition, air-blast dried cells of S. cerevisiae D8, M12, S13; H. uvarum S6; and I. orientalis KMBL5774 entrapped in 2 % Ca-alginate beads and soaked in protective agents (10 % skim milk containing 10 % sucrose, 10 % raffinose, 10 % trehalose, 10 % trehalose, and 10 % glucose, respectively) after air-blast drying at 37 °C for 5 h showed 90, 87, 92, 90, and 87 % viability, respectively. All dried entrapped yeast cells showed survival rates of at least 51 % after storage at 4 °C for 3 months.     

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs

Summary The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3-azido-3-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mm) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent K _m and V _max revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest. Offprint requests to: J. Magdalou     

Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads

Alginate microbeads, produced by emulsion/internal gelation, were studied for the entrapment and microcultivation of microbial cells with biotechnological potential. An anaerobic consortium which was selected for its capacity to degrade complex carbohydrates, and a pure culture of cellulose degrading bacteria were used for entrapment studies. Optimization of conditions for the formation of spherical alginate microbeads in sizes between 20 and 80 μm were examined. The best conditions were achieved by combining rapeseed methyl ester as oil phase and stirring at 100 rpm using a rotation impeller. Calcium alginate microbeads produced under these conditions were shown to present morphological stability, with large pores in the internal matrix that favours microcolony development. Finally, single cells were observed inside the beads after the entrapment procedure and microcolony formation was confirmed after cultivation in cellobiose.     

Biodegradation of naphthalene by free and alginate entrapped Pseudomonas sp

Naphthalene degradation by freely suspended and immobilized cells of Pseudomonas sp. isolated from contaminated effluents has been investigated in batch cultures and continuously in a packed bed reactor. Naphthalene concentration was varied from 25 mM to 75 mM, the temperature (30 degrees C) and pH (7.0) were kept constant. The results showed good acclimation of the strain to carbon source and degradation rate was highly affected by initial concentration. Alginate-entrapped cells have given good yields although initial rates were not as high as those encountered with free cells. A first order exponential decay kinetic model was proposed with values of parameters for each initial concentration. A laboratory scale packed-bed bioreactor was designed using parameters calculated above and continuous experiments were realized at different flow rates (100 to 200 ml/h), with different feed concentrations and operating during 30 days. The conversion at low feed concentrations and low flow rates was complete whereas at high flow rates and high concentrations it was less efficient because of diffusional limitations and short residence time.     

Production of alginate beads by a rotative atomizer

Summary The use of a rotative flat disc atomizer for the production of biocatalysts immobilized in alginate gel was investigated. The influence of viscosity (0.28 to 1.53 Pa · s), disc diameter (5 to 20 cm), flow rate (0.5 to 2 L/min) and rotational speed (1.6 to 5 rev/s) on drop formation mechanisms, bead diameter and their distributions were investigated. Bead diameters ranging from 1 to 3 mm were been obtained. Flow rate and viscosity increases resulted in higher bead diameters but increasing disc diameter and rotational speed had a decreasing effect. A viscosity of 0.65 Pa · s would prove best for production of beads with good size distribution and mechanical strength. Predictive equations for the mean volumetric bead diameter were determined.     

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