Immobilized Cell Preparations Research Articles

Effects of Constant Electric Field on Biodegradation of Phenol by Free and Immobilized Cells of Bradyrhizobium japonicum 273

It is shown that bacteria Bradyrhizobium japonicum 273 were capable of degrading phenol at moderate concentrations either in a free cell culture or by immobilized cells on granulated activated carbon particles. The amount of degraded phenol was greater in an immobilized cell preparation than in a free culture. The application of a constant electric field during cultivation led to enhanced phenol biodegradation in a free culture and in immobilized cells on granulated activated carbon. The highest phenol removal efficiency was observed for an anode potential of 1.0 V/S.H.E. The effect was better pronounced in a free culture. The enzyme activities of free cells for phenol oxidation and benzene ring cleavage were very sensitive to the anode potential in the first two steps of the metabolic pathway of phenol biodegradation catalyzed by phenol hydroxylase—catechol-1,2-dioxygenase and catechol-2,3-dioxygenase. It was observed that at an anode potential of 0.8 V/S.H.E., the meta-pathway of cleavage of the benzene ring catalyzed by catechol-2,3-dioxygenase became competitive with the ortho-pathway, catalyzed by catechol-1,2-dioxygenase. The obtained results showed that the positive effect of constant electric field on phenol biodegradation was rather due to electric stimulation of enzyme activity than electrochemical anode oxidation.

Production of dihydroxyacetone from an aqueous solution of glycerol in the reaction catalyzed by an immobilized cell preparation of acetic acid bacteria Gluconobacter oxydans ATCC 621

Dihydroxyacetone (1,3-dihydroxy-2-propanone, DHA) is applied in the food and cosmetic industries as well as in pharmacy and medicine. It is produced as a result of incomplete oxidation of glycerol by acetic acid bacteria Gluconobacter oxydans. This reaction is catalyzed by PQQ-dependent membrane-bound glycerol dehydrogenase. The research developed a method of obtaining DHA by oxidation of a 3 % aqueous solution of glycerol (pH 7.5) at a temperature of 23 °C, with the only reaction biocatalyst being an immobilized cell preparation obtained from G. oxydans cells. After 5 days of the process, DHA concentration in the solution accounted for 27.2 g/L and the reaction efficiency for 94 %. After 4 days of the reaction run in culture media with pH 5.0, at a temperature of 28 °C, free or immobilized cells of G. oxydans produced on average 25 g of DHA/L at the reaction efficiency of 87 %.

Enhanced production of δ-aminolevulinic acid, bilipigments, and antioxidants from tropical algae of India

We studied the enhanced production of high quality biomass, δ-aminolevulinic acid (δ-ALA), bilipigments, and antioxidants from five tropical blue green algae (cyanobacteria) in a full factorial design using free and immobilized cells in batch culture. Production of nutraceuticals was high in spray dried powder prepared from immobilized cell cultures. Nostochopsis lobatus showed superiority over rest of the species with respect to bilipigments, δ-ALA, nutritive value, antioxidant capacity, and ascorbate oxidase (APX) activity. Antioxidative capacity of phycobiliproteins extracted from these cyanobacteria (121.15 μM TE/g, Nostoc verrucosum to 217.62 μM TE/g, Nostochopsis lobatus) was invariably higher than those observed for higher plant sources and substantially increased under immobilized cell culture condition. Antioxidative enzyme, ascorbate oxidase remained stable in dry food preparations with considerably high activity under immobilized cell preparations (APXmax, 3.40 μmol/min/mg chlorophyll). These observations have important connotations in light of upcoming food and nutraceutical industries in the global market. Use of immobilized cells in batch culture could be an effective approach for scaling up production for commercial use.

Fermentation of banana media by using κ-carrageenan immobilized Lactobacillus acidophilus

Fermentation media were prepared by using banana as the raw material, and cell immobilization of Lactobacillus acidophilus by κ-carrageenan entrapment was applied to enhance the fermentation efficiency. Gel beads of diameters around 3.0 mm were prepared for the immobilized cells, ripe bananas were used for preparation of media, and both free and immobilized cells were employed to carry out the fermentation for 80 h. Cells leaked out from the gel beads and proliferated in the medium solution during the fermentation of immobilized cells. The final viable cell number reached 10 5 CFU/ml in the medium suspension, over 10 8 CFU/(ml gel) in gel beads for the immobilized cell fermentation and around 10 6 CFU/ml for the free cell fermentation. Immobilized cells withstand the adverse conditions in banana media resulting in better fermentation efficiency compared to free cells. Variation of fructooligosaccharides (FOS) in banana media was not significant in immobilized cell fermentation compared to free cell. Immobilized L. acidophilus fermented banana medium was found to possess synbiotic properties.

Effect of matrix on ( S)- p-chlorodiphenylmethanol production by immobilized Debaryomyces marama

The product ( S)- p-chlorodiphenylmethanol is obtained through stereoselective reduction of p-chlorobenzophenone using Debaryomyces marama cells, immobilized on Raschig rings. This process involved continuous fermentations in bioreactors for a period of 10 days. The conversion was 86% with a volumetric productivity of 0.248 litre −1 d −1. Comparative experiments with D. marama cells, incorporated in alginate, k-carrageenan or agar–agar gels were also performed. These results demonstrated superiority of the immobilization on ceramic carriers compared to other methods. Passive immobilization of D. marama cells on ceramic carriers has many advantages in comparison with gel-incorporation of the cells. For example, during a long bioreactor run, only a limited amount of cells could remain incorporated within the gel. Therefore immobilized cell preparations will be a convenient methodology in the production of optically active compounds such as pharmaceutical drugs.

Marennine production by agar-entrapped Haslea ostrearia Simonsen

The pennate Diatom Haslea ostrearia was successfully immobilized in agar-gel disks and tested for marennine production in batch experiments. The concentration of pigment recovered varied between 34 and 92 mg l −1 liquid medium, according to the initial cell loading of the gel structures, which corresponded to volumetric production rates between 15 and 50 mg day −1 l −1 gel. Average specific productivities of immobilized cells were higher than those of free cells. Immobilized-cell preparations could be used three times (working time > 1000 h) without significant loss in production efficiency.

Production of optically pureL-alanine by immobilized Pseudomonas sp. BA2 cells

The conditions for immobilizing the new L-aminoacylase-producing bacterial strain, Pseudomonas sp. BA2, by entrapment in κ-carrageenan gel, were investigated. The optimal gel concentration and cell load were determined. The addition of CoCl2 and N-acetyl-L-alanine to the immobilizing matrix enhanced L-aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated. Enzyme activity in immobilized cells was optimal at a pH of 6·5 using 0·15 mol dm−3 Tris–maleate buffer at 45°C. The presence of 0·7 mmol dm−3 CoCl2 in the enzymatic reaction mixture improved L-aminoacylase activity. The immobilized cell preparation was used for the production of L-alanine from N-acetyl-DL-alanine in a batch reactor. Conversions of 100% were obtained using substrate concentrations ranging from 20 to 200 mmol dm−3. The reactor production was 0·74 mol h−1 g cell−1 dm−3 which is noticeably higher than that previously reported in the literature. © 1998 Society of Chemical Industry

Stabilization studies of l-aminoacylase-producing Pseudomonas sp. BA2 immobilized in calcium alginate gel

Pseudomonas sp. BA2 showing l-aminoacylase activity was immobilized by entrapment in calcium alginate gel. To enhance operational stability, the immobilized cells were treated with hardening reagents using four different methods: treatment of the cells with glutaraldehyde before immobilization, stabilization of the immobilized cell preparation with glutaraldehyde or glutaraldehyde and hexamethylenediamine, and finally, activation of the alginate solution with periodate followed by treatment with polyethyleneimine. Very active and stable immobilized cell preparations were obtained when Pseudomonas sp. BA2 cells immobilized in calcium alginate were treated with 20 m m glutaraldehyde. Compared with the untreated preparation, the enzymatic activity was markedly increased (132%) and the half-life period was increased by 4 h. Moreover, the suitability of the biocatalyst for reuse in batch processes was noticeably improved (only 60% of the initial l-aminoacylase activity was lost after four runs). When the immobilized and stabilized cell preparation was used for l-alanine production in a continuous stirred-tank reactor, substrate conversion at the reactor outlet was higher than that obtained with the untreated beads. The reactor production after 33 h was 61 of 17 m m l-alanine solution (9 g optically pure amino acid) more than double that obtained using the untreated immobilized cells, and significantly higher than those previously reported in the literature.

Lipolytic activity in free and immobilized cells of Phoma glomerata

AbstractThe lipolytic activity of free and immobilized whole cells of the pathogenic fungus Phoma glomerata was demonstrated, and several properties of the lipase involved were determined. Free fungal cells and small pieces of immobilized cells, prepared by spontaneous colonization on a solid surface or entrapped in calcium alginate, were incubated with triolein in buffered medium. Different incubation conditions were assayed to optimize the reaction, to determine the effects of heating and time on stability of the immobilized preparations and the time course of the reactions. Although the enzyme cleaves all ester bonds of triolein, it shows some preference for the outer bonds. An optimal pH of 7.5–8.0, optimal temperature of 45°C for free and 50°C for immobilized cell preparations, the necessity for substrate emulsifiers, and reaction independence from calcium and magnesium were demonstrated. Results suggest that immobilized whole cells of P. glomerata would be a suitable tool to study its lipid physiology and to explore further the possible biotechnological use of its lipase activity.

Immobilization of Pseudomonas sp. BA2 by entrapment in calcium alginate and its application for the production of l-alanine

The conditions for immobilizing the new l-aminoacylase-producing bacterial strain Pseudomonas sp. BA2 by entrapment in calcium alginate gel were investigated. The optimal gel concentration and cell loading were determined. It was demonstrated that the addition of the substrate n-acetyl- l-alanine to the immobilizing matrix enhanced l-aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated to ascertain which conditions were suitable for the enzymatic reaction. Optimal pH, temperature, and concentration of Tris-maleate buffer were determined. The influence of adding CoCl 2 on the enzymatic reaction rate was studied and the optimal concentration of the activator was determined. Stability studies showed that the immobilized cell preparation is not adequate for use in repeated batch processes. Continuous operation in a stirred tank reactor allowed us to determine the biocatalyst half-life (7 h approximately) but, due to the high l-aminoacylase activity, the reactor productivity (24.5 mmol of l-alanine in 8 h) was noticeably higher than that previously obtained in a packed bed reactor with Aspergillus ochraceus pellets. The results reported in this paper show the potential for using the immobilized Pseudomonas sp. BA2 in calcium alginate to produce l-alanine; however, before large scale production can be undertaken, further biocatalyst stabilization studies have to be made.

Conversion of d-sorbit to l-sorbose by cells of Acetobacter suboxydans immobilized in sintered glass

A simple method for immobilizing cells of Acetobacter suboxydans by adsorption on inorganic sintered glass carriers is described. The immobilized cell preparations exhibited 100% of the initial activity when converting d-sorbit to l-sorbose. This sintered glass was used in a fixed-bed loop reactor with a working volume of 0.2 l for semicontinuous and continuous experiments. A prolonged working life span was achieved, which could possibly satisfy requirements for scaled-up operations.

Design of Ca-alginate immobilized yeast cell beads with controlled low density to enhance their fluidization behaviour in bioreactors

The incorporation of small glass hollow spheres into Ca-alginate beads allows the density of immobilized cell preparations to be decreased. For immobilized yeast, glass hollow spheres at 14% (v/v) decrease the density from 1088 to 996 kg m-3, thus greatly changing their hydrodynamic properties. As a consequence, the operation of a three-phase fluidized bed reactor is clearly improved.

NADH production from NAD + with a formate dehydrogenase system involving immobilized cells of a methylotrophic Arthrobacter strain

A convenient and economical method of NADH production from NAD + has been established using a formate dehydrogenase system involving immobilized cells of a methanol-utilizing bacterium, Arthrobacter sp, KM62. Four kinds of cell entrapment were studied. An immobilized cell preparation showing a high NADH production activity was obtained by entrapment in a κ-carrageenan gel lattice. The NADH-producing activity of the immobilized cells was investigated under various conditions. The NADH-producing activity was evoked on the addition of Triton X-100 to the reaction mixture. The conditions for the continuous production of NADH with an immobilized cell column were also investigated. When a reaction mixture containing 10 μmol (6.63 mg) ml −1 NAD + was passed through the column (1.2 × 20 cm) containing 1.62 g (as dry weight) of immobilized cells, at a space velocity of 0.125 at 35°C, complete conversion was attained.

Effect of antibiotics on lysine production in free and immobilized cells of Bacillus subtilis

In immobilized cell preparations growth of cells outside the immobilization matrix as free cells is normally undesirable due to the appearance of cells in the product stream and clogging of such systems. Antibiotics could be used to arrest such free cell growth while allowing the synthesis and excretion of the product into the medium. Chloramphenicol (200 μ/ml) and/or novobiocin (10 μ/ml), when added during the growth of Bacillus subtilis allowed the production and excretion of lysine into the medium. Chloramphenicol at 200 μ/ml effectively arrested free cell growth and hence the lysine being produced was almost entirely due to immobilized cells. Novobiocin on the other hand at concentrations of 100 μ/ml, stopped free cell growth, but also prevented the production of l-lysine. Productivities and yields of lysine were adversely affected by chloramphenicol or novobiocin, probably due to a great decrease in cell viability.

Fructose production by immobilizedArthrobacter cells

ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×109 CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.

Use of immobilized microbial cells for accelerated maturation of beer

Immobilized cell preparations of various bacteria able to reduce diacetyl to butane 2,3-diol have been tested under conditions appropriate for use in the accelerated removal of the diketone from potable alcoholic beverages. Strains ofBacillus polymyxa that are sufficiently active when incubated anaerobically at 5°C and pH 4 have been identified.

Synthesis of l-dopa by escherichia intermedia cells immobilized in a polyacrylamide gel

Escherichia intermedia cells were immobilized by entrapment in a polyacrylamide gel and used for l-dopa synthesis from pyrocatechol, pyruvate and ammonia. An immobilized cell preparation containing 75 mg cells/g gel retained 45%–50% of the activity of free cells. The effect of temperature, pH and substrate concentration of the initial rate of l-dopa synthesis was very similar for free and immobilized cells. Substrate inhibition was observed for pyrocatechol, pyruvate and ammonia. In a batch reactor, 5.4 g·l-1l-dopa was obtained, with 100% conversion yield of pyrocatechol and l-dopa productivity of 0.18 g·l-1·h-1. The use of a pyrocatechol-borate complex decreased by-product formation and catalyst inactivation.

Stabilization of κ‐carrageenan gel with polymeric amines: Use of immobilized cells as biocatalysts at elevated temperatures

Spherical beads of kappa-carrageenan containing entrapped cells were prepared in a two-step process. First, the beads were formed by dispersing a warm carrageenan cell suspension into stirring oil. After cooling (gelation) the beads were cured by treatment with amines. Ten amines of various sizes and structures were tested. We evaluated the mechanical strength and the applicability of aminetreated gels as immobilization matrices. The results of critical compression tests indicate that linear and branched polyethylenimines (PEI) are both good curing agents. PEI treated carrageenan beads also exhibited superior resistance to heat and abrasion. Furthermore, PEI polymers were demonstrated to be effective in stabilizing the lactase activity of the free and immobilized Bacillus stearothermophilus cells. The immobilized cell preparations of Saccharomyces cerevisiae, B. stearothermophilus, and Flavobacterium sp. were treated with branched PEI and evaluated for the activity of invertase (EC 3.2.1.26), lactase (EC 3.2.1.23), and glucose isomerase (EC 5.3.1.18), respectively, in a packed bed reactor at 60 degrees C. The apparent half-lives were 108, 39, and 64 days, respectively.

Biotransformation of 3‐methoxy‐8,14‐seco‐1,3,5(10),9(11) estratetraen‐14,17‐dione (secodione) to its 17β‐hydroxy derivative (secol) using immobilized yeast cells (Pichia farinosa Y‐118)

The yeast cells of Pichia farinosa Y-118 were immobilized in polyacrylamide gel and used for 17 beta-oxidoreduction of secondione to secol. The loss of hydroxysteroid oxidoreductase activity of cells was found to be insignificant during immobilization. The preparation exhibited greater temperature stability as compared to free cells. The ratio of reaction volume to the volume of immobilized biocatalyst in the range 1.4-1.9 was found to be satisfactory for the reaction conditions studied. This ratio played a significant role in the stability of the catalyst particle, since beyond a critical value the disintegration of gel granules was rapid resulting in sharp decline of activity. The immobilized cell preparation could be used 50 times over a period of 100 days without loss of activity. However, the activity declined in further reuses, leaving the preparation 50 and 35% active after its 60th and 70th uses, respectively.

Maintenance and operational stability of immobilized Arthrobacter simplex for the ▵ 1-dehydrogenation of steroids

The stability of the ▵ 1-dehydrogenation system of Arthrobacter simplex immobilized in calcium alginate has been studied. A high stability was related to the ability of the cells to utilize a carbon source such as d-glucose or steroid. Inhibition of de novo protein synthesis reduced the ▵ 1-dehydrogenase [3-oxosteroid: acceptor) ▵ 1-oxidoreductase, EC 1.3.99.4] stability of the immobilized cells. The operational stability of immobilized cell preparations in the presence of the steroid degradation inhibitor, α,α-dipyridyl, could not be improved significantly by supplementing steroid substrate suspensions with either d-glucose or yeast extract.