Free Cell System Research Articles

Ethanol production from non-sterilized beet molasses by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from non-sterilized beet molasses by free and immobilized S. cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum ethanol concentration (53 g/litre) at an initial sugar concentration of 250 g/litre and F = 250 ml/h. In the free cells system, the maximum ethanol productivity (3.8 g/litre h) was obtained at a substrate concentration of 250 g/litre and F = 500 ml/h with 30.6% ethanol yield and 81% sugars utilization. In the immobilized cells system, a maximum ethanol productivity of 3.5 g/litre h was achieved at an initial sugar concentration of 250 g/litre and F = 500 ml/h with 31.2% ethanol yield and 73.3% sugars utilization. In repeated fed-batch culture, immobilized S. cerevisiae cells gave higher overall ethanol concentration compared to the free cells. In this case, the immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 8 days.

Continuous decolorization and AOX reduction of bleach plant effluents by free and immobilizedTrametes versicolor

Abstract Continuous decolorization of bleach plant effluents was studied using white‐rot fungus, Trametes (Coriolus) versicolor. The experiments were carried out using free and immobilized fungus. In the free cell system, 56–77% continuous decolorization and 41–69% AOX reduction were achieved with a retention time of 24 hours. Treatment of bleach plant effluents with the same fungus encapsulated in calcium alginate polymer beads resulted in 76–83% decolorization and 43–59% AOX reduction. These are the highest levels achieved in a continuous bioreactor system using Trametes (Coriolus) versicolor.

On the kinetics and effectiveness of immobilized whole-cell batch cultures

A model for growth-associated fermentations is presented for batch cultures of immobilized whole cells. The model encompasses exponential and steady-state growth phases and the effectiveness of the biocatalyst. Based on the model equations, experimental methods have been developed for the determination of the kinetic parameters: maximum specific growth rate (μ max ), Monod constant ( K m ) and effectiveness factor (η). These methods are illustrated by ethanol fermentations using Saccharomyces cerevisiae cells entrapped in alginate beads. The kinetic parameters obtained show consistency with those determined for free-cell systems.

On the kinetics and effectiveness of immobilized whole-cell batch cultures

A model for growth-associated fermentations is presented for batch cultures of immobilized whole cells. The model encompasses exponential and steady-state growth phases and the effectiveness of the biocatalyst. Based on the model equations, experimental methods have been developed for the determination of the kinetic parameters: maximum specific growth rate (μmax), Monod constant (Km) and effectiveness factor (η). These methods are illustrated by ethanol fermentations using Saccharomyces cerevisiae cells entrapped in alginate beads. The kinetic parameters obtained show consistency with those determined for free-cell systems.

Improvement of plasmid stability of recombinantBacillus-subtiliscells in continuous immobilized cultures

The immobilization of recombinant Bacillus subtilis in K-carrageenan gel beads has been performed in order to study the growth conditions inside the gel beads and to improve plasmid stability. Bacterial colonies showing high cell density were studied using scanning electron microscopy. A series of continuous cultures of free and immobilized B. subtilis MT119 (pHV1431, pIL252 and pIL252 Kpn) have been developed without selection pressure. In the free-cell systems, it was found that a loss of plasmid vectors occurred after a short period. In contrast, in the immobilized cell systems, plasmid-free segregants were not detected in any of the cases during the first 80 h of the culture.

Production of a bacteriocin active on lactate-fermenting clostridia byLactococcus lactissubsp.lactisimmobilized in coated alginate beads

SummaryWe report the isolation and immobilization of a nisinogenic strain (NZ1) ofLactococcus lactissubsp.lactis, active on gas-forming lactate-fermenting clostridia responsible for late blowing of Asiago and Montasio cheeses. The bacteriocin (nisin) produced by strain NZ1 is pronase-sensitive and is released in culture media during the growth phase. Using the sensitive indicator strainLactobacillus delbrueckiisubsp.bulgaricusNCDO 1489, a rapid microtitre plate based assay was developed for quantitative determination of the bacteriocin produced by NZ1 cells, either free or immobilized in gel beads. Scanning electron microscopy of cells immobilized in calcium alginate coated beads and viable counts of the surrounding medium showed that no cell leakage occurred during a 24 h assay. The bacteriocin released from immobilized cells reached, after 5 and 24 h, concentrations comparable to that of the free cell system after 3–4 h incubation in culture media.

Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-column bioreactor.

Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells. Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42,000 units (U).1(-1)] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.01 h-1 with high volumetric productivity (450 U.1-1.h-1) and yield.

Biological treatment of phenolic wastes: Comparison between free and immobilized cell systems

Phenol degradation by free and immobilized cells ofFusarium flocciferum was studied in a chemostat at steady-state conditions. For the free cell system the dilution rates varied from 0.02 to 0.13h−1, with a total phenol removal up to 0.08h−1. Wash-out seemed to set in at 0.11h−1. The immobilized cells showed virtually complete phenol utilization at 1g/l, over a period of four months. At D=0.2h−1 and above 1g/l phenol, the complete phenol removal is not achieved: a progressive increase in the outlet concentration was observed attaining a value of 284mg/l at 1.5g/l.

Thermus 2S from Thai hot springs: isolation and immobilization

Three strains of thermophilic bacteria producing extracellular protease have been isolated from hot springs in Chiang Mai, Thailand. The bacterium producing the highest amount of protease has been selected and identified as belonging to the genusThermus, and is tentatively calledThermus 2S. The isolate is a Gram-negative, rod shaped bacterium. It exhibited maximum growth around 60°C at pH 7. Entrapment of the microbial cells in calcium alginate maintained the cell viability. Protease production from immobilized cells using 2 g wet cells per 10 ml 3% (w/v) sodium alginate was higher than that from a free-cell system using 2% inoculum.

Promotive action of ceramics on yeast ethanol production, and its relationship to pH, glycerol and alcohol dehydrogenase activity

A yeast strain, Saccharomyces cerevisiae KPY32 isolated from pito, a traditional West-African alcoholic beverage, was immobilized in porous ceramic beads as a means of improving its ethanol production. Stationary fermentation cultures at different temperatures were made using semi-synthetic medium and fermentation parameters including ethanol production, sugar consumption, cell growth and pH were monitored. Glycerol production, and the activity of alcohol dehydrogenase (ADH) of the various systems were monitored. It was found that immobilization of the yeast resulted in improved ethanol production, at conversion rates above 93% of the theoretical value. The pH of the immobilized systems was also stabilized at around 4.0, glycerol production was higher, and the ADH activities were higher than those of free-cell systems. Ethanol production at the high temperature of 37° C was also improved by immobilization. The promotive action was found to be related to the pH, presence of glycerol and the enhancement of ADH activity.

Degradation of Phenol by Immobilized Mycelium of Fusarium Flocciferum in Continuous Culture

Continuous phenol degradation was studied in a completely mixed reactor, at steady state conditions, using the mycelium of Fusarium Floociferum immobilized in Polyurethane foam. Experiments at different dilution rates, 0.069, 0.118 and 0.196h−1 with an influent phenol concentration of 1g/l, showed a virtually complete substrate utilization over a period of four months. Low biomass productivity values of suspended cells were found, 0.21, 0.34 and 1.81g d.w./l. day, especially when compared with those obtained with the free cell system at similar conditions: 0.955 and 6.73g d.w./l.day for 0.063 and 0.116h dilution rates. The immobilized cells behaviour at different feed substrate concentrations was studied at D=0.196h.−1 corresponding to a retention time of 5.1h. The results indicate that above 1g/l the complete phenol removal is not achieved. A progressive increase in the outlet concentration was observed, attaining a value of 284mg/l at 1.5g/l. Respiratory experiments made with the immobilized cells taken from the reactor along with these experiment, indicate that the viability is retained by the mycelium, pointing out its ruling part in the phenol degradation. Culture fluid viscosity measurements were made and the rheological characteristics of the two systems were investigated. A non-Newtonian fluid was observed in the free cell system, with a shear-thinning behaviour. The immobilization had a beneficial effect on the rheology of the fluid i.e. the viscosity was much lower,1.2 c.p.s. and there was a tendency to approach Newtonian behaviour.

Production of propionic acid from whey ultrafiltrate by immobilized cells of Propionibacterium shermanii in batch process

Whole cells of Propionibacterium shermanii were immobilised on an inert support to produce propionic acid from cheese whey ultrafiltrate in a batch process. A maximum concentration of 11·5 g litre −1 propionic acid was obtained in the immobilized-cell system against 2·25 g litre −1 propionic acid in the free-cell system over the same time of incubation. The addition of CaCO 3 increased the acid production rate, the final concentration of the product and the final yield of acid per unit weight of lactose consumed. Yeast extract had a significant effect on the final concentration and the final yield of the acid per unit of substrate consumed but not on the product formation rate. The addition of K 2HPO 4 did not improve significantly the production of propionic acid.

Comparative studies of physiological and environmental effects on the production of cyclosporin A in suspended and immobilized cells of Tolypocladium inflatum

Process improvement of the production of cyclosporin A (Cy A), a powerful immunosuppressive fungal metabolite, has been undertaken by analyzing suspended and immobilized cell cultures in parallel. Conidiospores of the producer microorganism, Tolypocladium inflatum, were entrapped into porous celite particles. Easier germination of the entrapped spores and more active growth of the immobilized cells were manifested when compared with free cell cultures initiated with spores or with mycelial inocula. Significant differences in precursor flow between the immobilized and free cell systems were evident when the effects of L-valine (a constituent amino acid of the Cy A molecule) on Cy A biosynthesis were compared in the two systems. For the freely suspended cells, L-valine supplemented early in the fermentation served as a possible precursor or stimulator of Cy A biosynthesis. A significant increase in specific production and Cy A yield on carbon source was observed in this system relative to suspended cultures supplemented with L-valine during or after exponential growth. In contrast to the free cell cultures, the addition of L-valine during the initial stage of immobilized cell growth had a negative effect on Cy A production but resulted in somewhat increased cell growth. This suggests an incompatibility between primary and secondary metabolic networks involved in Cy A biosynthesis in the immobilized state upon external addition of the amino acid.

A comparative study of cadmium uptake by free and immobilized cells from activated sludge

Abstract Cadmium biosorption by free and immobilized cells from activated sludge was studied in batch experiments to determine the effects of immobilization on the biosorption properties of the microorganisms. The method of immobilization consisted of the entrapment of the microorganisms in calcium alginate beads. In a first step, the time course of cadmium uptake and the effects of pH and temperature on biosorption were studied in each case. Then the biosorption pattern and the effect of biosorbent concentration were investigated. It was found that the magnitudes of the effects of pH and temperature on biosorption were reduced by the entrapment of the cells, but the major effects of the entrapment consisted of a very high reduction of the biosorption rate and a modification of the biosorption pattern. On the one hand, cadmium biosorption by free cells followed the pattern of the Freundlich isotherm over wide ranges of cell and cadmium concentrations in solution. On the other hand, biosorption by the beads could be described by the Freundlich isotherm only when bead concentration was kept constant because the parameters K and n varied with bead concentration. Finally, it was found that, as expected, the entrapment of the cells significantly reduced their intrinsic biosorption capacity due to a loss of available binding sites. For cases where biosorption is the primary objective, free cell systems should therefore be preferred to entrapped cell processes to remove cadmium from aqueous effluents by using activated sludge. Biosorption isotherms show that cadmium can be biosorbed up to more than 20,000 times above water concentrations utilizing free cells at 30 °C and pH 6.6. However, the numerous advantages of immobilized cells in reactor operation should also be considered to select the appropriate technology.

Immobilization of genetically engineered cells: a new strategy for higher stability

The r-DNA clones improve the bioprocess and provide better economics, if and when properly developed. In recent times, many approaches were made to improve the stability of recombinants in a reactor which includes both genetic and environmental methods, but many of them were proved to be unsuccessful in the scale-up process. The immobilization technique, exploited recently for the cultivation of recombinants, in many cases gave high cell concentrations, better expression of cloned gene products and also maintained plasmid stability for longer periods in a host under continuous operation in comparison to a free cell system. Many plasmids and hosts were tested for improved stabilities. So far, no explanation was provided for higher stability in the immobilized system. However, it was observed to reduce the competition between the plasmid harboring and plasmid free cells in a matrix. The stability of recombinant strains under immobilization technique is affected by various factors, and these are important parameters for the commercial process. Thus, the immobilization system is promising for the successful cultivation and scale-up of genetically engineered cells.

Mercury accumulation and volatilization in immobilized algal cell systems

Rapid removal of mercury from growth medium and its uptake by free and alginate-entrappedChlorella has been observed. Immobilized cell systems accumulated more mercury than free cell systems. In addition, both volatilized significant quantities of mercury. Studies show, however, that mercury lost in this way may re-enter the aqueous phase and subsequently be accumulated by immobilized cells.

Characterization of an Immobilized Biocatalyst System for Production of Thermostable Amylase

AbstractAn immobilized biocatalyst system for enzyme production using a thermophilic organism is described. Bacillus stearothermophilus (ATCC 29609) immobilized on Rohm and Haas IRA–938 ion exchange resin was utilized for the production of a highly thermostable α‐amylase in a bench scale airlift fermentor. The equilibrium between suspended bacterial cells and the resin fit a Langmuir‐type sorption isotherm. Cell and α‐amylase productivity data for free cell and immobilized cell continuous culture were compared as functions of the dilution rate. Cell growth rates for free and immobilized cells were consistent with the Monod kinetic model, α‐Amylase production for free cells was described by an unstructured model associated with cell growth. The immobilized cell systems gave higher α‐amylase productivity than the free cell system but did not achieve the productivities predicted by the unstructured kinetic model.

Immobilization of Tolypocladium inflatum spores into porous celite beads for cyclosporine production

Conidia of Tolypocladium inflatum were immobilized into porous celite beads for the production of the immunosuppressive agent cyclosporine (cyclosporin A, Cy). The strong enhancement in volumetric Cy production as well as specific production based on unit cell mass was demonstrated in immobilized cell culture as compared with freely suspended culture. Altered metabolic pattern in the immobilized fungus was indicated by the production of pink-colored pigments. In view of the diffusional resistance typically encountered in immobilized cell culture, bead size limitation was investigated. Cells entrapped into small beads (diameter in the range of 150 μm–207 μm) showed markedly higher and sustained productivity compared with cells entrapped into middle-sized (208–294 μm) or large-sized (295–500 μm) celite particles. The immunosuppressant yields on carbon or nitrogen source for the immobilized cell culture were up to three-fold higher than for the free cell culture, thus clearly indicating that the immobilized cell system provides more efficient utilization of substrates for the biosynthesis of Cy than the free cell system, especially when the fungal cells are entrapped into small sized celite particles.

Increased stability of pBR322-related plasmids inEscherichia coliW3101 grown in carrageenan gel beads

The stability and the copy number of pBR322, pBR325 and pBR328 were studied during continous cultures of free and immobilized E. coli W3101 without selective pressure. In the free-cell system, it was found that pBR328 and pBR325-free E. coli cells appeared after a lag period. They rapidly overgrew the cultures and the plasmid copy number subsequently declined. On the other hand, an increase in the proportion of pBR322- carrying cells during a free continuous culture was observed. This increase correlated with that of plasmid copy number. By contrast, in the immobilized- cell system, plasmid free segregants were not detected in all the cases even after 250 generations. We have also shown that plasmid copy number remained constant and phenomena such as fluctuations or genetic modifications which occured after long term growth of bacteria in a free continuous culture could be avoided throughout cell immobilization.

Continuous production of small peptides from milk proteins by extracellular proteases of free and immobilized Serratia marcescens cells

The continuous production of small peptides from buttermilk protein proteolysis has been studied using whole free and immobilized Serratia marcescens cells as an enzyme source. The hydrolysis reaction was carried out in a reactor at various dilution rates from 0.15 to 0.50 h −1. Results showed that the cell population was stable, even at high flow rates, but the extent of hydrolysis varied as a function of residence time, and decreased as the dilution rate increased. Data from the distribution of the resulting peptides according to molecular weight and aromatic residues showed that the hydrolysates obtained result from the common action of an endopeptidase system favored by low flow rate, and an exopeptidase system that preferentially hydrolysed on aromatic terminal peptide and that was favored by high flow rate. In both reactors, the large protein residues were hydrolysed extensively, but more free amino acids were liberated in the immobilized cell system than in the free cell system. We obtained the best production of useful peptides corresponding to the characteristics of a dietary product in the free cell system, at a dilution rate of 0.25 h −1 ( 57% peptides with 2–10 amino acid residues in the hydrolysate).