Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was deomonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels

Immobilized microbial cells have been used extensively in various industrial and scientific endeavours. However, immobilized cells have not been used widely for environmental applications. This review examines many of the scientific and technical aspects involved in using immobilized microbial cells in environmental applications, with a particular focus on cells encapsulated in biopolymer gels. Some advantages and limitations of using immobilized cells in bioreactor studies are also discussed.

Environmental applications of immobilized microbial cells: A review

Publisher Summary In order to be suitable for technological applications, catalysts should be stable under operational conditions for weeks or months. With continuous research it is found that enzymes can be stabilized against thermal inactivation. There are three methods that can be employed in the attempt to make enzymes more thermostable: immobilization, chemical modification, and inclusion of additives. Using these methods, rate constants of thermo inactivation of many enzymes have been reduced by as much as 103–105 times; there are enzymes that even without any stabilization display remarkable thermal resistance. For example, Bacillus stearothemphilus a-amylase retains 90% of its activity after 1 hour at 90°C. It is found that at 100°C in 0.1 N HCI, the halflife of adenylate kinase exceeds 30 minutes. Bacillus lichenifomnis amylase continuously operates at 100–115°C. The aforementioned enzymes are made of the same building blocks as other, far less thermostable enzymes. Developments in protein chemistry and the understanding of thermophily, along with sensible analyses of enzyme thermoinactivation and use of common sense, will undoubtedly lead to many new approaches to stabilization of enzymes at high temperatures.

Stabilization of enzymes against thermal inactivation.

Enzyme stabilization by immobilization

Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023

The kinetics of the enzymatic hydrolysis of sucrose by invertase have been examined, with particular emphasis on high substrate concentration. Initial rates of reaction were determined by following the production of glucose directly as a function of time over a wide range of substrate concentrations (0.04M to 2.06M). The resulting data reveal a reaction rate that increases gradually until the sucrose concentration reaches about 0.29M, after which the reaction velocity decreases with increasing sucrose concentration. Previous workers (e.g., Nelson and Schubert1) have reported a peak reaction velocity as determined by indirect polarimetric measurements of glucose, at a sucrose concentration of about 0.17M. These measurements, however, neglect the intermediate oligosaccharides formed by the transferase action of invertase,8–10 and assume equal amounts of glucose and fructose. According to Anderson et al.,10 these oligosaccharides interfere by producing an erroneously low reaction rate. Experimental results of this work confirm Anderson's observations, and show a further reaction rate increase of nearly 20% between sucrose concentrations of 0.177M and 0.285M under the same conditions of temperature, pH, and enzyme-concentration.



Effects of substrate diffusion, solution viscisity, water concentration, and substrate inhibition were experimentally studied and the results incorporated into a kinetic model that has proven satisfactory in modeling the experimental results. This model takes into account inhibition by primary substrate, with concentration of the secondary substrate water, as a rate limiting factor at sucrose concentrations greater than 0.285M.



The effects of the mixing, in terms of volumetric power input, on the relation rate have been tested. Approximately 40-fold increase in volumetric power input caused on increase in the reaction rate. These experiments have shown that bulk mass transfer is not a rate limiting factor under the experimental conditions.

Kinetic modeling of the hydrolysis of sucrose by invertase

Immobilization of whole cells in a membraneous form.

Stable and active derivatives of collagen-immobilised whole microbial cells containing glucose isomerase have been successfully prepared. The degree of crosslinking, swelling behaviour, and hydrophile/hydrophobe ratio of the complex was examined, since these factors could have a strong influence on the diffusional characteristics of the membrane. The studies on efficiency of glutaraldehyde tanning and its effect on the activity of the immobilised cells provided the connecting link between the network properties and the mass transfer effects. The catalytic pathway for the reversible enzymatic conversion of glucose to fructose has been elucidated by developing a kinetic model capable of describing the observed data over the entire range of substrate concentrations. In arriving at this model, we have also found that the bulk phase mass transfer limitations are negligible. Regarding the internal transport resistance, it has been determined that the effectiveness factor is between 0.95 and 1. A reactor design equation incorporating activity, packing density, and contact efficiency correction factors has been developed. Based on this equation, computer simulations of continuous reactor operation were performed to generate concentration–space time profiles. The model was verified experimentally and a close agreement between the predicted and observed values corroborated the correctness of the proposed bioreaction mechanism, the rate expression, and the design equation. The kinetics of a competing side reaction were found to be too slow to influence significantly the overall results.

Reaction kinetics and mass transfer in glucose isomerisation with collagen‐immobilised whole microbial cells

Enzymatically active Protein-whole microbial cell complex membranes suitable for effecting enzymatic reactions are prepared by forming a dispersion containing synthetic polypeptide or natural protein macromolecules and whole microbial cells, casting a membrane from the dispersion and drying the membrane. In an alternative embodiment, the membranes are formed by electro-codeposition from a dispersion containing the macromolecules and cells. It is desirable to pre-treat the microbial cells to destroy proteolytic activity of the cells to prevent the cells from autolyzing and from destroying the membrane.

Rakesh Saini

Papers

Kinetic modeling of the hydrolysis of sucrose by invertase

Immobilization of whole cells in a membraneous form.

Reaction kinetics and mass transfer in glucose isomerisation with collagen‐immobilised whole microbial cells

Preparation of protein membranes containing microbial cells