Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

Production of proteoglycans by immobilized chondrocytes: Effect of divalent cations

Summary Rabbit articular chondrocytes immobilized in alginate beads maintained normal morphology and metabolic activity (glucose consumption) for more than two weeks whether calcium, barium, or strontium were used for the gel forming. Only Ca- and Ba-alginate immobilized chondrocytes produced proteoglycans in the external medium. Only Ca-alginate beads produced proteoglycans at a constant rate.     

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.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Zymomonas mobilis

The production of ethanol from starch by a coimmobilized mixed culture system of aerobic and anaerobic microorganisms in Ca-alginate gel beads was investigated. The mold Aspergillus awamori was used as an aerobic amylolytic microorganism and an anaerobic bacterium, Zymomonas mobilis, as an ethanol producer. By controlling the mixing ratio of the microorganisms in the inoculum size, a desirable coimmobilized mixed culture system, in which the aerobic mycelia grew on and near the oxygen-rich surface of the gel beads while the anaerobic bacterial cells mainly grew in the oxygen-deficient central part of the gel beads, was naturally established under the aerobic culture conditions, and ethanol could be directly produced from starch by the system. The ethanol productivity by the system in flask culture was particularly affected by the shear stress (dependent on the shaking speed) which controlled the mycelial growth on the surface of the gel beads. Under optimum culture conditions in the flask culture, the glucose produced was instantly consumed, and was not observed in the culture broth; the final concentration of ethanol produced from 100 g/L starch was 25 g/L and the yield coefficient for ethanol, Y(pls), was 0.38. The ethanol productivity by the coimmobilized mixed culture system was compared with those by other various culture systems and the advantages of the system were clarified.     

Reductive dechlorination of perchloroethylene and the role of methanogens

Abstract Perchloroethylene (PCE) was reductively dechlorinated to trichloroethylene in a 10% anaerobic sewage sludge. About 80% of the initially added PCE (300 nmol) was dechlorinated within three weeks. The calculated rates were 250 nM and 445 nM · day⁻¹ during the first and second weeks of incubation, respectively. The depletion of PCE varied in sludges obtained from different sources.
The role of methanogenesis in the dechlorination of PCE was evaluated by inhibiting the methanogens by addition of bromoethane sulfonic acid, a potent methanogenic inhibitor. Dechlorination of PCE was significantly inhibited in sludges amended with the inhibitor. Almost 41–48% less PCE was dechlorinated in sludges containing 5 mM BESA, indicating a relation between the two processes (methanogenesis and dechlorination). Direct proof that methanogens can transform chlorinated aliphatic compounds was obtained using axenic cultures of acetate-cleaving methanogens. Methanosarcina sp , originally isolated from a chlorophenol degrading consortium, showed significantly higher dechlorinating activity as compared to Ms. mazei . Based on these studies and other recently reported observations, it appears that methanogens/methanogenesis play an important role in the anaerobic dechlorination of chlorinated aliphatics such as PCE.     

Transport and consumption rate of O2 in alginate gel beads entrapping hepatocytes

Experiments carried out with hepatocytes entrapped in alginate gel particles with a cell density of about 10⁷ cells ml^–1 showed an oxygen consumption rate of about 2.2×10^–16 mol cell^–1 s^–1. Under these conditions, no inner anoxic core is present in 1.8 mm diameter beads. From these data, the maximum bead size consistent with non-critical O₂ concentration at the bead center has been evaluated for different cell densities and O₂ concentrations in the medium.     

Effect of immobilization on the stability ofClaviceps purpurea protoplasts

Summary Protoplasts released with high efficiency from vegetative and productive hyphae ofClaviceps purpurea were immobilized in 2% Ca-alginate. The yield of active immobilized protoplasts depended upon the age of the mycelium from which protoplasts were derived and was found to be 25–43% in comparison with native hyphae. During incubation in a modified production medium immobilized protoplasts were stable for at least 10–12 days. No external growth of regenerated hyphae from spherical beads of alginate gel with entrapped protoplasts was observed for 13–15 days of the batchwise incubation.     

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.     

Degradation of 4-aminobenzenesulfonate by alginate encapsulated cells of Agrobacterium sp. PNS-1

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.     

Development of a microchannel emulsification process for pancreatic beta cell encapsulation

In this study, we developed a high-throughput microchannel emulsification process to encapsulate pancreatic beta cells in monodisperse alginate beads. The process builds on a stirred emulsification and internal gelation method previously adapted to pancreatic cell encapsulation. Alginate bead production was achieved by flowing a 0.5–2.5% alginate solution with cells and CaCO₃ across a 1-mm thick polytetrafluoroethylene plate with 700 × 200 μm rectangular straight-through channels. Alginate beads ranging from 1.5–3 mm in diameter were obtained at production rates exceeding 140 mL/hr per microchannel. Compared to the stirred emulsification process, the microchannel emulsification beads had a narrower size distribution and demonstrated enhanced compressive burst strength. Both microchannel and stirred emulsification beads exhibited homogeneous profiles of 0.7% alginate concentration using an initial alginate solution concentration of 1.5%. Encapsulated beta cell viability of 89 ± 2% based on live/dead staining was achieved by minimizing the bead residence time in the acidified organic phase fluid. Microchannel emulsification is a promising method for clinical-scale pancreatic beta cell encapsulation as well as other applications in the pharmaceutical, food, and cosmetic industries.     

Enhancing the Viability of Lactobacillus plantarum Inoculum by Immobilizing the Cells in Calcium-Alginate Beads Incorporating Cryoprotectants

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in determining viability. The rate of rehydration and the volume of fluid used have been identified as two important factors. One possible means of controlling these is by immobilizing the cells before lyophilization within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the microenvironment within the two bead types was probably different. The protective effect of the bead microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.     

Anaerobic bacteria that dechlorinate perchloroethene

In this study, we identified specific cultures of anaerobic bacteria that dechlorinate perchlorethene (PCE). The bacteria that significantly dechlorinated PCE were strain DCB-1, an obligate anaerobe previously shown to dechlorinate chlorobenzoate, and two strains of Methanosarcina. The rate of PCE dechlorination by DCB-1 compared favorably with reported rates of trichloroethene bio-oxidation by methanotrophs. Even higher PCE dechlorination rates were achieved when DCB-1 was grown in a methanogenic consortium.     

Anaerobic bacteria that dechlorinate perchloroethene.

In this study, we identified specific cultures of anaerobic bacteria that dechlorinate perchlorethene (PCE). The bacteria that significantly dechlorinated PCE were strain DCB-1, an obligate anaerobe previously shown to dechlorinate chlorobenzoate, and two strains of Methanosarcina. The rate of PCE dechlorination by DCB-1 compared favorably with reported rates of trichloroethene bio-oxidation by methanotrophs. Even higher PCE dechlorination rates were achieved when DCB-1 was grown in a methanogenic consortium.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Optimization of alpha-amylase immobilization in calcium alginate beads

alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C.     

Biological sulfide oxidation using autotrophic Thiobacillus sp.: evaluation of different immobilization methods and bioreactors

Aims:  Evaluation of various immobilization methods and bioreactors for sulfide oxidation using Thiobacillus sp. was studied.
Methods and Results:  Ca-alginate, K-carrageenan and agar gel matrices (entrapment) and polyurethane foam and granular activated carbon (adsorption) efficacy was tested for the sulfide oxidation and biomass leakage using immobilized Thiobacillus sp. Maximum sulfide oxidation of 96% was achieved with alginate matrix followed by K-carrageenan (88%). Different parameters viz. alginate concentration (1%, 2%, 3%, 4% and 5%), CaCl₂ concentration (1%, 2%, 3%, 4% and 5%), bead diameter (1, 2, 3, 4 and 5 mm), and curing time (1, 3, 6, 12 and 18 h) were studied for optimal immobilization conditions. Repeated batch experiments were carried out to test reusability of Ca-alginate immobilized beads for sulfide oxidation in stirred tank reactor and fluidized bed reactor (FBR) at different sulfide concentrations.
Conclusions:  The results proved to be promising for sulfide oxidation using Ca-alginate gel matrix immobilized Thiobacillus sp. for better sulfide oxidation with less biomass leakage.
Significance and Impact of the Study:  Biological sulfide oxidation is gaining more importance because of its simple operation. Present investigations will help in successful design and operation of pilot and industrial level FBR for sulfide oxidation.     

Comparative study of methanol, butyrate, and hydrogen as electron donors for long-term dechlorination of tetrachloroethene in mixed anerobic cultures

This study examined the ability of different electron donors (i.e., hydrogen, methanol, butyrate, and yeast extract) to sustain long-term (500 days) reductive dechlorination of tetrachloroethene (PCE) in anerobic fill-and-draw bioreactors operated at 3:1 donor:PCE ratio (defined on a total-oxidation basis for the donor). Initially (i.e., until approximately day 80), the H(2)-fed bioreactor showed the best ability to completely dechlorinate the dosed PCE (0.5 mmol/L) to ethene whereas, in the presence of methanol, butyric acid or no electron donor added (but low-level yeast extract), dechlorination was limited by the fermentation of the organic substrates and in turn by H(2) availability. As the study progressed, the H(2)-fed reactor experienced a diminishing ability to dechlorinate, while more stable dechlorinating activity was maintained in the reactors that were fed organic donors. The initial diminished ability of the H(2)-fed reactor to dechlorinate (after about 100 days), could be partially explained in terms of increased competition for H(2) between dechlorinators and methanogens, whereas other factors such as growth-factor limitation and/or accumulation of toxic and/or inhibitory metabolites were shown to play a role for longer incubation periods (over 500 days). In spite of decreasing activity with time, the H(2)-fed reactor proved to be the most effective in PCE dechlorination: after about 500 days, more than 65% of the added PCE was dechlorinated to ethene in the H(2)-fed reactor, versus 36%, 22%, and <1% in the methanol-fed, butyrate-fed, and control reactors, respectively.     

Real and pseudo oxygen gradients in Ca-alginate beads monitored during polarographic Po2-measurements using Pt-needle microelectrodes

Polarographic microcoaxial needle electrodes were used to measure internal profiles of dissolved oxygen tension (Po(2)) within single Ca-alginate beads of different diameter containing entrapped cells of Saccharomyces cerevisiae. For the investigations, single beads coming from variable growing conditions and distinct cultivation stages were fixed in a special holding device. In dependence on microbial growth steep oxygen gradients were observed. The Oxygen penetration depth at steady state lay between 50 and 100 mum. After 8 h of cultivation time, the anaerobic space within the beads (phi 2 mm; cultivation in a packed bed reactor) is beginning at approximately 130 mum, whereas the anaerobic space within the beads (phi 2 mm) coming from the shaker flask culture is located approximately 440 mum below the bead surface. Surprisingly, steep gradients were also observed, when recording profiles from cell-free Ca-alginate beads of different diameter and alginate concentrations. The steep oxygen gradients apparently had to be interpreted as pseudo-Po(2)-gradients. These results were borne by several effects, such as formation of artifacts and diffusion barriers in front of the electrode tip or oxygen "availability" at the tip and consumption of oxygen by the electrode itself. These phenomena could be documented by microscopic observation and photography. Thus, to obtain real Po(2)-profiles it is important to be exactly informed about the physical, chemical, and biological properties of the material to be investigated. Furthermore, it is necessary to apply a special stepwise puncture technique with distinct step-in/step-out movements of the electrode: e.g., unidirectional or contradirectional puncture techniques. (c) 1994 John Wiley & Sons, Inc.     

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell-free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from10⁵ cfu ml⁻¹ to 10³ cfu ml⁻¹ after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.     

Role of calcium alginate and mannitol in protecting bifidobacterium

Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (a(w)) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl(2) solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The a(w) of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without a(w) adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low a(w)s. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low a(w)s. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C).