Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Microbial growth in a fixed volume: studies with entrapped Escherichia coli

Summary Stresses exerted by a growing entrapped colony of Escherichia coli up to 3 atmospheres were measured by incorporating a pressure transducer into a specially designed immobilized cell reactor. This stress is comparable in magnitude to the turgor pressure generated by Gram negative bacteria. In complementary experiments, cell densities as high as 850 grams dry weight per liter were measured in aggregates of starved E. coli subjected to controlled applied stresses up to 9 atmospheres. Cell volume reduction was quantitatively described by a model which incorporated the fundamental osmotic properties of the cell. compression of entrapped cells was qualitatively corroborated by electron microscopic examination. These results suggest that entrapped growing bacteria can exert a substantial stress on their surroundings and that dewatering of the starved cell population in an entrapped system may occur. Both of these consequences of entrapped cell growth can be understood in terms of the osmotic behavior of the cells.     

Entrapment of Lavandula vera cells with synthetic resin prepolymers and its application to pigment production

Summary Cultured cells of Lavandula vera were entrapped with photosensitive synthetic resin prepolymers (PVA-SbQ). PVA-SbQ-entrapped cells grew well inside gel matrices and synthesized de novo blue pigments in the presence of l-cysteine as an inducer. The entrapped cells were superior to calcium alginate-entrapped cells judging from cell growth and total pigment productivity. Release of the pigments, which were almost insoluble in water, from the gels was markedly enhanced by the increase in hydrophilicity of the cell-entrapping gels. The entrapped cells could be used repeatedly for the pigment production.Dedicated to Professor Dr. Georg Manecke on occasion of his 70th birthday     

Biodegradation of dichloroacetic acid by entrapped and adsorptive immobilized Xanthobacter autotrophicus GJ10

The degradation of dichloroacetic acid (DCA) by free, Ca-alginate entrapped and adsorptive immobilized cells of Xanthobacter autotrophicus GJ10 has been studied in various experimental systems. Entrapped cells tolerated increasing concentrations of DCA better than free cells. Free and adsorptive immobilized cells degraded DCA most effectively at maximum O₂ supply, 34°C and an initial pH value of 8.0. The degradation of high DCA concentrations led to a decrease in the pH value and to a stagnation of mineralization, particularly with free or entrapped cells. Due to the stabilization of pH, the supplementation of acetate or succinate resulted in a complete degradation of higher DCA concentrations. Higher degradation rates than in shake cultures were achieved in air-bubble and packed-bed fermentors. DCA was mineralized faster by free or entrapped X. autotrophicus GJ10 than by adsorptive immobilized cells, which, however, were able to remove higher DCA concentrations. The results of the recent investigations with immobilized X. autotrophicus GJ10 are an important prerequisite for the application of this bacterium in waste treatment systems. Correspondence to: U. Heinze     

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summary dl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.Dedicated to the 65th birthday od Professor Dr. G. Manecke     

Immobilized catalase-containing yeast cells: Preparation and enzymatic properties

The cell of Saccharomyces cerevisiae previously induced for catalase (EC 1.11.1.6) activity were immobilized by entrapment of intact cells in acrylamide polymerized by γ irradiation (100 kR). Yeast cells showed an enhancement in catalase activity on entrapment, an effect similar to that observed on treatment with organic solvents like toluene. The cells pretreated with toluene, however, showed complete loss of catalase activity on entrapment. The entrapped enzyme exhibited a narrow pH optimum, reduced K_m for H₂O₂, and a decrease in thermostability. The temperature optimum of catalase was also decreased from 60 to 40°C on immobilization. A tenfold decrease in the activation energy was also observed. The enzyme in the entrapped cells was, however, stable toward inactivation by γ irradiation. Unlike the intact cells, the entrapped yeast cells did not have the ability to induce catalase.     

Stability of calcium-alginate during citric acid production of immobilized Aspergillus niger

Summary This article introduces an easy to handle immobilization apparatus for the entrapment of microbial cells, organelles and enzymes in spherical gel beads.Ca-alginate beads with entrapped cells of Aspergillus niger showed typical shrinking behaviour (from 3.00 mm to 2.25 mm particle diameter). A loss of stability down to 20% of the initial strength during precultivation of the fungus and within the following citric acid production occurred. The observed particle shrinkage was due to the increasing acidification of the medium, whereas the decreased mechanical strength was caused by the entrapped growing microorganism. This was confirmed by electron scanning micrographs, indicating a sponge-like gel structure within the region of enhanced mycelium growth which reduced diffusional resistance of the matrix. Therefore no differences were found between citric acid production of Ca-alginate entrapped Aspergillus niger at 3 mm and 1.5 mm initial particle size.     

Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.

The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.

The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.

Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 days) provided the accumulation of indole on the fibres was avoided.     

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.     

Permeabilization of cultivated plant cells by electroporation for release of intracellularly stored secondary products

Summary Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm^–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.     

Bioconversion of bi- and tri-cyclic monophenols by alginate-entrapped cells of Mucuna pruriens L. and by the partially purified Mucuna-phenoloxidase

Although alginate-entrapped cells of Mucuna pruriens L. possess a low substrate specificity, only para-substituted monocyclic phenols have been ortho-hydroxylated into catechols so far. In this study, compounds with more complex chemical structures were found to be substrates using entrapped cells of M. pruriens as well as the partially purified Mucuna-phenoloxidase. Thus, 5-, 6- and 7-hydroxylated 2-aminotetralins and a tricyclic compound, 9-hydroxy N-n-propyl hexahydronaphthoxazine, were converted into catechols. After isolation using preparative HPLC, the identity of the products was confirmed by MS. In general, for the entrapped cells and the enzyme preparation identical substrate specificities were found.This publication is dedicated to the memory of Prof. Alan S. Horn, Ph.D., who deceased at January 2, 1990     

Laboratory-scale production of lignin-degrading enzymes by free and entrapped cells ofPhanerochœte chrysosporium in a tubular air-lift bioreactor

The production of lignin-degrading enzymes by free and entrapped cells ofPhanerochœte chrysosporium in a tubular air-lift bioreactor was studied. Under optimized cultural conditions the production of lignin peroxidase by free cells, calcium-alginate-entrapped cells and scouring-mesh-entrapped cells was in a ratio of 520∶720∶950 mU/mL, while the production of manganese peroxidase was in a ratio of 350∶480∶620 mU/mL. The stability of the entrapped cells by fed-batch systems was highest after 3 feeding experiments which is similarly demonstrated in the repeated use of the preparations in batch system.     

Degradation of 2-chloroethanol by free and immobilized Pseudomonas putida US 2

The degradation of 2-chloroethanol by Pseudomonas putida US 2 was investigated in shaking flasks, air-bubble columns and packed-bed fermenters by free cells, calcium-alginate-entrapped cells and on cells on granular clay adsorbed. Entrapped cells tolerated increasing concentrations of 2-chloroethanol better than free cells. Their maximum degradative activity could be observed at 34°C and pH 7.0. The degradation of 2-chloroethanol leads to a decrease of pH and to a stagnation of mineralization, particularly with free or entrapped cells. Following the stabilization of pH, supplementation with succinate resulted in a complete degradation of higher 2-chloroethanol concentrations. Less 2-chloroethanol was degraded in air-bubble columns and larger amounts in packed-bed fermenters. 2-Chloroethanol was mineralized faster by free or entrapped P. putida US 2 than by adsorbed cells, which, on the other hand, were able to remove higher concentrations of the compound. The results with P. putida US 2 are a good indication that this microorganism could be used in waste-water treatment and soil-decontamination systems.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

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.     

Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.     

Immobilized Isochrysis galbana (Haptophyta) for long-term storage and applications for feed and water quality control in clam (Meretrix lusoria) cultures

The marine microalga Isochrysisgalbana was cultivated and entrapped inalginate beads for long-term storage. Theentrapped cells were alive and maintainedtheir physiological activities after oneyear of storage in absolute darkness at4 ^°C without a liquid medium. Thenumber of cells in the beads increased morethan 32 times when they were subsequentlyre-cultured in an aqueous medium for fiveweeks, showing that they had remained aliveduring storage. TEM observations showedthat the entrapped cells reduced their cellcovering and pyrenoid size compared withthe normal free-living cells afterlong-term storage. The algal beads werealso applied to feed and water qualitycontrol in clam cultures' leading to amarked decrease in ammonium concentrations.Algal cells escaped from the beads provideda food source for the clams. This mightreduce the cost of clam culture compared totraditional culture methods. Therefore,immobilized I. galbana can be usedfor long-term preservation of algal stockin the laboratory and applied practicallyto clam cultures.     

Behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel and performing alcoholic fermentation

Summary The behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel was studied during their continuous function in an ethanol-producing reactor. Polymerization destroys 40% to 80% of the cells, depending on their physiological state. A three day adaptation phase is required before ethanol production stabilizes and this phase corresponds to an increase in cell concentration in the gels and to protein synthesis. The amounts of DNA, glucan, glycogen and trehalose are different in entrapped and free cells. Microscopic observation shows that 75% to 85% of the cells lose their integrity and that the remainder appear to multiply normally. Within a gel particle, both viability and fermentation activity are heterogeneous. A high percentage of cells have low viability and low fermentation activity. A proportion of cells remains capable of forming colonies and these cells have higher fermentation activity and are preferentially localized at the surface of gel particles.     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

Immobilization of microbial cells in crosslinked,prepolymerized, linear polyacrylamide gels: Antibiotic production by immobilized Streptomyces clavuligerus cells

A mild new method for the immobilization whole microbial cells has been developed. Cells were suspended in a solution of preformed, linear, water-soluble Polyacrylamide chains, partially substituted with acylhydrazide groups. The Prepolymerized backbone polymer was crosslinked, in the presence of viable cells, by stoichiometric amounts of dialdehydes such as glyoxal, glutardialdehyde, and period ate-oxidized polyvinyl alcohol. The crosslinking reaction, carried out in cold, neutral physiological conditions resulted in cells entrapped in gels with physical properties similar to those of the common Polyacrylamide gels. However, cell damage generally caused by the acrylamide monomer was avoided. Resting Streptomyces clavuligerus cells, possessing a high capacity for antibiotic production, were entrapped according to this procedure. These immobilized cells produced cephalosporins continuously for 96 h with yields similar to those of free resting cells. The same cells, when immobilized by direct polymerization acrylamide monomers, yielded significantly lower amount of antibiotics.