Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Characterization of metabolically active developmental stage of Aspergillus niger cells immobilized in polyacrylamide gel

Summary The in-situ development of Aspergillus niger entrapped in polyacrylamide gel from spores and the gel surface characteristics were studied during the repeated shake flask batch citric acid fermentation. A marked increase in the rate of citric acid production was observed with the periodic replacement of culture with fresh media at an interval of 6 days reducing the fermentation time nearly to half. The metabolically active A. niger cells for citric acid production were characterized by the appearance of thick and bulbous hyphae scattered in and on the gel surface.     

Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l^− 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (q_s) and gluconate production (q_p) reduced progressively. Interestingly, gradual q_s and q_p reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.

     

Lactic acid fermentation by cells of Lactobacillus rhamnosus immobilized in polyacrylamide gel

Summary The process of lactic acid fermentation of lactose to lactic acid by Lactobacillus rhamnosus ATCC 7469 has been studied. The following processes have been explored: growth kinetics, as well as lactose utilization, production of lactic acid and further degradation of lactic acid. The immobilization experiments were conducted with microbial cells entrapped in polyacrylamide gels. Gels with different ratios of the monomer (acrylamide) and the cross-linking agent (N,N′-methylene-bis-acrylamide) have been tested. These were used in a repeat-batch process. The current processes inside and outside the gel particles were subjects of examination. The evolution of the activity of immobilized cells with repeated use showed that the particles served mainly as a donor of cells for the free culture. In all experiments a very high degree of conversion, 85–90% was observed. After several runs however, the particles were exhausted for microbial cells. A kinetic model of the process of lactic acid production was developed. This model allowed the evaluation of the effect of microbial growth and diffusion limitations inside the gel particles on the process rate and the separate contribution of the free and immobilized cells to the overall fermentation process upon multiple use.     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

Development of a fermentation method using immobilized cells under unsterile conditions. 1. Protection of immobilized cells against anti-microbial substances

A method of protecting immobilized cells against inhibitory substances in the fermentation medium was investigated with the aim of developing a process for fermentation under unsterile conditions. It was found that yeast cells could be protected against the inhibitory effects of p-hydroxybenzoic acid esters by co-immobilizing the cells with vegetable oils. In such a system, the cells grow only in the water phase of the gel beads where most components of the fermentation medium are retained. On the other hand, the p-hydroxybenzoate that diffuses into the gel beads is retained mainly in the oil phase of the beads. Consequently, the p-hydroxybenzoate concentration in the water phase remains too low to inhibit the metabolic activities of the immobilized cells. The effectiveness of a vegetable oil in protecting the immobilized cells against an inhibitory substance depends on the partition coefficient of the substance between the oil and water, the concentration of the oil and the initial cell concentration.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

2,3,5-triphenyltetrazolium chloride as a viability assay for immobilized plant cells

Grape (V. vinifera L. cv Gamay Fréaux) cells were entrapped in calcium alginate beads. The effect of bead diameter and gel concentration on the viability of the cells was checked. The reduction of 2,3,5-triphenyltetrazolium chloride as well as O₂ consumption were used as viability test, and their results compared. The existence of diffusional limitations for O₂ introduces an unaccuracy as high as 25% for the O₂ consumption method. The reduction assay is more simple, precise and reliable.     

Immobilized bacterial cells containing a thermostable β-galactosidase

Summary A constitutive -galactosidase has been localized in the cytosol of thermoacidophilic bacterium Caldariella acidophila. Cells have been entrapped in polyacrylamide gel with full retention of enzymic activity; no activity decrease is observed after 8 months of storage. Enzyme properties in entrapped cells are similar to those of the free enzyme. A 73% hydrolysis of lactose has been achieved in a continuous system on a 2 ml entrapped cell column operating at 70°C; half life in these conditions is 30 days.In this paper we report preliminary data on immobilization of cells of Caldariella acidophila, an extreme thermophilic bacterium having a constitutive -galactosidase (EC 3.2.1.23) activity.     

Entrapment of microbial cells within polyurethane hydrogel beads with the advantage of low toxicity

Summary A new method is described for the entrapment of microbial cells in polyurethane (PUR) hydrogel beads. This hydrogel is produced from a hydrophilic pre-polymer blocked with bisulphite by adjusting the pH between 4 and 6.5. Bisulphite-blocked isocyanate has a substantially lower toxicity against living cells than unblocked (conventional) isocyanates. The poly(carbamoylsulfonate) (PCS) hydrogels have optimal elastic properties and therefore can be used for a matrix of biocatalysts in an agitated reactor as well as in a fluid-bed reactor. The results of ethanol fermentation ofSaccharomyces cerevisiae entrapped in PCS hydrogel beads, and of the denitrification activity of immobilizedParacoccus denitrificans are promising. In contrast, entrapped cells in conventional PUR hydrogels didn’t show any activity.     

THE STUDY OF THE INFLUENCE OF TEMPERATURE AND INITIAL GLUCOSE CONCENTRATION ON THE FERMENTATION PROCESS IN THE PRESENCE OF Saccharomyces cerevisiae YEAST STRAIN IMMOBILIZED ON STARCH GELS BY REVERSED-FLOW GAS CHROMATOGRAPHY

The technique of reversed-flow gas chromatography (RFGC) was employed for the determination of the alcoholic fermentation phases and of kinetic parameters for free and immobilized cell systems, at different initial glucose concentrations and temperature values. In addition to this, due to its considerable advantages over other techniques, RFGC was used for the characterization of a new biocatalyst, yeast cells immobilized on starch gel, and especially wheat starch gel. Immobilization of wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels in order to prepare new biocatalysts with great interest for the fermentation industry. The RFGC led with great accuracy, resulting from a literature review, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. A maximum value of rate constants was observed at initial glucose concentration of 205 g/L, where a higher number of yeast cells was observed. The increase of glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constants were decreased. The decrease of fermentation temperature caused a substantial reduction in the viability of immobilized cells as well as in rate constant values. Activation energies of corn starch gel presented lower values than those of wheat starch gel. However, the two supports showed higher catalytic efficiency than free cell systems, proving that starch gels may act as a promoter of the catalytic activity of the yeast cells involved in the fermentation process.     

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     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

The use of immobilized cells to stabilize orsellinate depside hydrolase of Pseudevernia furfuracea

Mixed cells of Pseudevennia Lun-Lunacea thallus have been entrapped in polyacrylamide gel and used in a continuous column process to hydrolyze evernic acid. Equimolar amounts of both orsellinic and everninic acids are recovered. The activity of entrapped cells, which contain orsellinate depside hydrolase, is unaltered over a period of two months.     

Immobilization of plant protoplasts: Viability studies

Protoplasts of Daucus carota Ca68 and Catharanthus roseus have been immobilized by entrapment in gelforming polysaccharides (kappa-carrageenan, agarose and alginate). Uniform spherical beads of carrageenan and agarose containing the protoplasts have been prepared by utilizing an inert hydrophobic phase (vegetable oil). The entrapped protoplasts are viable and stabilized towards osmotic shock by the polymeric backbone. Standard methods have been used to study the viability and integrity of the entrapped protoplasts. Upon incubation in a relatively simple medium the immobilized protoplasts show a much higher viability after 14 days as compared to free protoplasts under the same conditions. The viability of D. carota protoplasts has also been monitored by an enzyme activity present in the cells (digitoxigenin 58-hydroxylase).     

Hollow fiber bioartificial liver utilizing collagen-entrapped porcine hepatocyte spheroids

A xenogeneic hollow fiber bioreactor utilizing collagen-entrapped dispersed hepatocytes has been developed as an extracorporeal bioartificial liver (BAL) for potential treatment of acute human fulminant hepatitis. Prolonged viability, enhanced liver-specific functions, and differentiated state have been observed in primary porcine hepatocytes cultivated as spheroids compared to dispersed hepatocytes plated on a monolayer. Entrapment of spheroids into the BAL can potentially improve performance over the existing device. Therefore, studies were conducted to evaluate the feasibility of utilizing spheroids as the functionally active component of our hybrid device. Confocal microscopy indicated high viability of spheroids entrapped into cylindrical collagen gel. Entrapment of spheroids alone into collagen gel showed reduced ability to contract collagen gel. By mixing spheroids with dispersed cells, the extent of collagen gel contraction was increased. Hepatocyte spheroids collagen-entrapped into BAL devices were maintained for over 9 days. Assessment of albumin synthesis and ureagenesis within a spheroid-entrapment BAL indicated higher or at least as high activity on a per-cell basis compared to a dispersed hepatocyte-entrapment BAL device. Clearance of 4-methylumbelliferone to its glucuronide was detected throughout the culture period as a marker of phase II conjugation activity. A spheroid-entrapment bioartificial liver warrants further studies for potential human therapy. (c) 1996 John Wiley & Sons, Inc.     

Alcoholic fermentation by immobilized yeast at high sugar concentrations

Summary Glucose fermentation bySaccharomyces cerevisiae immobilized by entrapment in agar, carrageenan, alginate and polyacrylamide gels, was compared to that of freely suspended cells at concentrations of 10–50% (w.w.) sugar. The rate of ethanol production by the entrapped cells was 20–25% higher than that of the free cells. Concentrations of up to 14,5% w/w ethanol (30% glucose initial concentration) could be obtained. A number of hypotheses for the improved alcoholic fermentation are discussed.     

Preparation and application of polymer-entrapped enzymes and microorganisms in microbial transformation processes with special reference to steroid 11-beta-hydroxylation and delta-dehydrogenation

Fungal cells from Curvularia lunata were entrapped in a crosslinked polyacrylamide gel. The gel-cells obtained as granules were applied in the microbial transformation of Reichstein compound S leading to cortisol through an 11-β-hydroxylation step. Some kinetic studies of this conversion using gel-cells were carried out. In addition, it was shown that gel-cell granules which had lost part of their 11-β-hydroxylase activity on storage could be reactivated yielding preparations with increased activity. From Corynebacterium simplex a steroid dehydrogenase catalyzing the Δ¹- dehydrogenation of cortisol leading to prednisolone was isolated and partially purified. The preparation was entrapped in a crosslinked polyacrylamide gel and the gel-enzyme granules obtained used in steroid dehydrogenation processes.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013