Immobilisation of cells in a linear polyacrylamide matrix: Degradation of a mixture of volatile fatty acids byAlcaligenes denitrificans.

Summary Cells of a potent volatile fatty acid degrading strain of the organismAlcaligences denitrificans were immobilised in a linear, prepolymerised, derivatised polyacrylamide gel, cross linked with glyoxal. The beads formed were structurally strong, and immobilised cells had an increased ability compared to an equivalent quantity of free cells, to degrade a mixture of volatile fatty acids at concentrations up to 6 g/l.     

Formation of trans-verbenol and verbenone from alpha-pinene catalysed by immobilised Picea abies cells

Both enantiomers and the racemate of alpha-pinene were transformed by Picea abies cells immobilised on alginate. The main products were cis- and trans-verbenol, the later being further transformed to verbenone. The enantiomeric purity of each product more or less corresponded to that of the substrate. Transformation by free cells was faster than that by the immobilised cells. The ratio of products differed to some extent between the transformation by free and immobilised cells.     

Optimum design of a series of continuous stirred tank reactors containing immobilised growing cells

The optimum design of a series of continuously operated stirred-tank reactors containing immobilised growing cells is described. Optimal design is defined as the minimal total holding time over the reactor series to achieve a certain degree of conversion. The analysis is made under the assumptions that there is a constant and equal concentration of immobilised biomass in all bioreactors along the series, no diffusion limitation takes place, all growth of the immobilised biomass will lead to an increase in suspended biomass, and that maintenance of the immobilised cells can be neglected. It is shown that the use of more than three bioreactors in series is likely to be de trop.     

Combined immobilized cultures producing fibrinolytic proteinases

The authors obtained combined immobilised systems composed of a culture producing fibrinolytic proteinases and a stimulating strain. The optimal ratio between the two cultures in gel was selected at a high starting cell density. Highly stable immobilised cultures were produced by growing the cells in gel particles. The interrelationship of the partners was studied in the binary immobilised culture. The biosynthetic activity of the system fell down to the level of a monoculture when the cells of the stimulating strain were eliminated from gel. The producing and stimulating strains are at equilibrium in associative immobilised cultures obtained by growing the cells in gel, and Arthrobacter is not eliminated. The mechanism of biosynthesis stimulation in a combined immobilised culture has been studied. Apparently, the procedure of immobilisation and the action of a stimulating compound exert the synergistic effect.     

Growth of immobilised plant cells in reticulate polyurethane foam matrices

Summary An inoculum of initially freely suspended cell aggregates ofCapsicum frutescens was immobilised in porous polyurethane foam matrices. Subsequent growth and substrate consumption of these immobilised cells in batch culture were measured and compared with those of suspension cultures. The results showed that the maximum specific growth rate of freely suspended cells was slightly higher than that of immobilised cells but the overall growth patterns and final cell yields were similar.     

Detoxification of hexavalent chromate by Amphibacillus sp. KSUCr3 cells immobilised in silica-coated magnetic alginate beads

Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe₃O₄) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe₃O₄ nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)₂, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca²⁺ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology.     

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.     

Use of polyurethane foam for the biodegradation of n-alkanes by immobilised cells of Pseudomonas

Pseudomonas strains C12B and A3 were separately immobilised in polyurethane foam and their ability to biodegrade n-decane or n-hexadecane tested and compared with that of freely suspended cells. In the presence of immobilised Pseudomonas C12B or A3 cells about 90% of n-alkane, originally present in liquid medium, was removed, 25%-40% of which was due to biodegradation.     

Plasmodium falciparum polypeptides interacting with human red cell membranes show high affinity binding to Band-3

P. falciparum proteins were labelled with [35S]methionine and harvested at various asexual stages. A number of parasite proteins bound to uninfected red cell membranes (ghosts). Some of these proteins differentially partitioned when ghosts were extracted with detergent. Several of these proteins bound very strongly to immobilised whole ghost proteins or immobilised purified Band-3 in a stage-specific manner, but not to a sham-coupled matrix or to immobilised Band-3 extract from cells rendered refractory to invasion. Such specific binding of parasite proteins to immobilised Band-3 supports recent conjecture as to its role as a host receptor during parasite invasion. However, our results demonstrate the complex and multifactorial nature of the interaction between parasite and host proteins during invasion and development.     

Studies on Cephalosporin-C production using immobilised cells of Cephalosporium acremonium in a packed bed reactor

The production of the β-lactam antibiotic, Cephalosporin-C in a packed bed bioreactor was studied using Cephalosporium acremonium immobilised in calcium alginate, bagasse, and silk sachets. The specific β-lactam antibiotic production rate of immobilised cells was about 120%, 125% and 133% at 100 h (free cells 100%) for calcium alginate, bagasse and silk sachets, respectively. A cells-to-carrier ratio of 3:2 was found to be optimum throughout these studies. The effect of recycle ratio indicated diffusion barriers to substrate transport.     

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl₂ concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl₂ concentrations higher than 0.4 M were utilised for bead preparation.     

Plasmodium falciparum polypeptides interacting with human red cell membranes show high affinity binding to Band-3

P. falciparum proteins were labelled with [³⁵S]methionine and harvested at various asexual stages. A number of parasite proteins bound to uninfected red cell membranes (ghosts). Some of these proteins differentially partitioned when ghosts were extracted with detergent. Several of these proteins bound very strongly to immobilised whole ghost proteins or immobilised purified Band-3 in a stage-specific manner, but not to a sham-coupled matrix or to immobilised Band-3 extract from cells rendered refractory to invasion. Such specific binding of parasite proteins to immobilised Band-3 supports recent conjecture as to its role as a host receptor during parasite invasion. However, our results demonstrate the complex and multifactorial nature of the interaction between parasite and host proteins during invasion and development.     

The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production

Hydrogen gas can be produced electrochemically by leading a current through two electrodes immersed in a NaCl solution. Bacteriorhodopsin (BR) a protein found in the purple membrane of Halobacterium halobium, is known to pump protons across the membrane upon illumination. In this study, the effect of BR on photoelectrochemical hydrogen production was investigated. A batch type bio-photoelectrochemical reactor was designed and constructed. The photoelectrochemical hydrogen production experiments were performed with free H. halobium packed cells or immobilised H. halobium cells. The cells were either immobilised in polyacrylamide gel (PAG) or on cellulose acetate membrane (CAM). Experiments were also performed with purple membrane fragments of H. halobium immobilised on cellulose acetate membrane. It was found that the presence of bacteriorhodopsin (BR) in the reactor enhances the hydrogen production rate upon illumination. Immobilisation increased the amount of hydrogen produced per mole of BR. Compared to control experiments without BR, the power requirement of the photoelectrochemical reactor per amount of hydrogen produced decreased fourfold when purple membrane fragments immobilised on CAM were used. The presence of BR regulates the pH of the system, increases the hydrogen production rate and causes light-induced proton dissociation, which lowers the electrical power requirement for the electrochemical conversion.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Product shifting by controlling medium pH in immobilised Streptomyces coelicolor A3(2) culture

Actinorhodin production by Streptomyces coelicolor A3(2) immobilised naturally in a porous support material was investigated in a 20 l bioreactor. The effect of pH-control on actinorhodin fermentation was conducted by performing different strategies. The combination of pH-control and immobilisation of the cells produced a different result than pH-control with freely suspended cells. The control of pH at 7.2 in an immobilised cell system altered the physiology of the cells shifting the product, actinorhodin, to another pigmented secondary metabolite, undecylprodigiosin. Although the cells were biologically active in such an environment, they were unable to produce actinorhodin. In the freely suspended cell system, however, actinorhodin biosynthesis was not hindered by controlling the medium pH.     

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.     

On the merits of viable-cell immobilisation

Many advantages have been claimed over the years for the use of immobilised cells, both as enzyme systems and as whole viable cell systems for complete fermentation reactions. However, few of the claims have been fully substantiated, and may not even be entirely justified. Most research is involved with single applications and the best that can be hoped for is some evidence that immobilised cells in each of these individual cases display some advantage over the equivalent free cell system. The purpose of this review is to assess the merits of viable cell immobilisation in the light of published literature and to elucidate the underlying mechanisms. Particular attention is paid to the generally unanticipated, but widely observed enhanced stability of immobilised cell fermentation processes.     

Studies on the accumulation of heavy metal elements in biological systems

Summary Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl₂, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl₂ over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.     

Artificial immobilization of the two-membered bacterial system Bdellovibrio bacteriovorus 109D--Escherichia coli B in polyacrylamide gel

The interaction of a parasite with a host was studied in the two-membered bacterial system, Bdellovibrio bacteriovorus 109D and Escherichia coli B, immobilised in polyacrylamide gel (PAAG). The parasite localised inside the host cells was found to be more resistant to the toxic action of PAAG components than free B. bacteriovorus. The latter lost its mobility and was inactivated in the matrix of the carrier whereas the intracellular parasite had a normal cycle of development in the periplasm of the infected cells. The dynamics of B. bacteriovorus and E. coli incidence in the liquid phase and in PAAG granules was studied while the immobilised system was incubated. The interaction in the immobilised system could be intensified by growing more bacterial host cells in PAAG particles. The immobilisation was shown to favour the survival of the parasite and the host in the two-membered system.     

Yeast Catalysed Reduction of β-Keto Esters (2): Optimisation of the Stereospecific Reduction by Zygosaccharomyces Rouxii

Zygosaccharomyces rouxii catalysed the reduction of ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) to the corresponding (S)-hydroxy ester (ethyl (S)-4-chloro-3-hydroxybutanoate) in high enantiomeric excess. The productivity of non-immobilised cells was compared to cells immobilised on a range of organic and inorganic supports. Cells immobilised in calcium alginate displayed a catalytic activity significantly higher than that of non-immobilised cells. A time dependent fall in the enantiomeric purity of the product was observed with the use of this matrix. This phenomenon was not seen in the reduction catalysed by non-immobilised cells.