Immobilized Cell Biocatalyst Research Articles

Optimization of Biocatalyst Specific Activity for Glycolic Acid Production

AbstractA chemoenzymatic process has been developed that employs an immobilized microbial nitrilase biocatalyst for the conversion of glycolonitrile to high‐purity glycolic acid. The specific activity of this immobilized cell biocatalyst decreased significantly during initial use in either consecutive batch reactions with catalyst recycle, or in a continuous stirred‐tank reactor, but the nitrilase activity remaining after this initial decrease was stable under the reactions conditions. The initial stability of this immobilized cell nitrilase catalyst has been improved by treatment of the microbial cells with glutaraldehyde prior to immobilization. Conditions for glutaraldehyde treatment were defined that completely inactivated the culture without significantly affecting nitrilase activity. A method for dehydration, storage and rehydration of the carrageenan‐immobilized cells has also been demonstrated that further improves the specific activity of this biocatalyst.

Use of immobilized cell biocatalysts in baking

Baking using baker's yeast immobilized in a starch–gluten–milk matrix (traditional fermented cereal food trahanas), containing viable lactic acid bacteria (LAB), and kefir (natural co-culture of yeasts and LAB) immobilized on orange peel, were investigated. The use of immobilized cells increased shelf life, delayed staling, and improved overall the quality of bread, compared with the traditional baker's yeast bread. These improvements were attributed to the reduction of pH, the lower moisture loss rates, and the presence of LAB, which are known to exhibit antimould properties. Better results were obtained using the sourdough method compared to the straight dough bread-making method. Headspace SPME GC–MS analysis showed that the use of immobilized cells increased the number of bread aroma volatiles, especially esters. The best results, including shelf life and overall bread quality, were obtained in the case of baker's yeast immobilized on trahanas, although kefir immobilized on orange peel seems to be a more cost effective biocatalyst.

Glucose production from maltodextrins employing a thermophilic immobilized cell biocatalyst in a packed-bed reactor

This research is focused on the development of an immobilized cell biocatalyst applicable for the conversion of maltodextrins into glucose at high temperatures. In particular, Escherichia coli BL21 (DE3) cells expressing α-glucosidase from Sulfolobus solfataricus MT4 were entrapped in a Ca-alginate polysaccharidic matrix obtaining over 70% immobilization yield. Compared to the free α-glucosidase, the immobilized cells showed a shift of optimal pH towards alkaline (from 5.0 to 5.5) and the same increasing activity up to 100 °C. These biocatalyst beads were packed into a tubular reactor, which was exploited for the biotransformation of a commercial dextrin mixture at industrial concentrations (30–40%, w/v) into glucose at 75 °C, achieving up to 98% conversion. The corresponding glucose productivity was about 1.75 g h −1 g biocatalyst −1.

Reaction Kinetics of Immobilized-Cell Denitrification. I: Background and Model Development

A simplified, quasi-steady-state model has been formulated for groundwater denitrification using immobilized cells. The model takes into account the diffusion-limited penetration of nitrate and nitrite into the immobilized-cell biocatalyst particle, uniform intraparticle cell density, equivalent slab geometry for the spherical particle, zero-order intrinsic reaction kinetics of the immobilized cells, sequential reduction of nitrate to nitrite and nitrite to dinitrogen within the particle, no inhibition of the sequential reactions by either nitrate or nitrite, and ideal plug flow conditions in the bioreactor. As a result, the reaction in the bulk fluid of the plug-flow bioreactor can be described by a half-order kinetics, and the process is characterized by the half-order reaction rate constants for nitrate and nitrite reduction. These constants incorporate the intrinsic reaction kinetics of the immobilized cells, the size and packing density of biocatalyst particles in the reactor, and the effective diffusion coefficients of nitrate and nitrite in the particle matrix.

A diffusion model and optimal cell loading for immobilized cell biocatalysts.

A diffusion model based on the random pore model is derived for immobilized cell biocatalysts and verified with 19 sets of experimental diffusion data. The predicted effective diffusivity relative to that for the support matrix reflects a quadratic dependence on the cell loading and contains a single parameter that depends on the intracellular diffusivity and the chemical partitioning coefficient. The model is used to predict optimal cell loadings that maximize the total reaction rate in an immobilized cell biocatalyst. A rule of thumb based on the diffusion model is obtained to the effect that the cell loading should be at least (1/3) for single reactions regardless of the kinetics and diffusional resistances. A means of calculating improved lower bounds is provided for cases where the cellular diffusional resistance is known but the kinetics are not. The optimal cell loadings for reversible first-order and for Michaelis-Menten kinetics are presented and demonstrated to be within the range of conditions of practical interest.

Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influence of diffusion in biological reaction systems. The broad substrate specificity and hydrolytic efficiency of OPH coupled with the ability to genetically engineer the enzyme for specific target OP neurotoxins enhance the suitability of OPH-based technologies for detoxification of these compounds. Cryoimmobilization provides a suitable vehicle as a cost-effective, efficient technology for bioremediation of environmental media contaminated with OP compounds.

Effect of oxygen limitations on monoclonal antibody production by immobilized hybridoma cells.

The productivity of an immobilized cell biocatalyst is often limited by the amount of oxygen that reaches cells located at interior regions of the biocatalyst. These diffusive limitations depend on a multitude of factors including the oxygen supply, the cellular uptake kinetics, and the cell density of the material. Large cell densities, which are desired for high productivity, are also likely to reduce the percentage of cells that receive an adequate supply of oxygen. To develop a better understanding of how different conditions affect biocatalyst behavior, a computational model of immobilized hybridoma cells was developed. The model accounts for oxygen diffusion and consumption, cell proliferation and death, and monoclonal antibody production. This model assumes that cellular productivity is limited only by the supply of oxygen and that the growth media is continually replenished so that nutrient levels remain high and wastes are eliminated. Biocatalyst performance is evaluated by monitoring the amount of monoclonal antibody produced by the cells. Model predictions agree with experimental measurements reported in the literature and indicate that for long operation time the supply of oxygen, biocatalyst size, and cell kinetics have a significant effect on biocatalyst performance, whereas the initial cell loading has only a relatively small effect. Under typical culture conditions, we find that oxygen penetrates to a maximum depth of about 0.4 mm. Accordingly, cells immobilized farther than this threshold distance receive an insufficient supply of oxygen.

Effect of Co-immobilization of Microporous Particles on the Overall Reaction Rate of Immobilized Cell Biocatalysts.

Cell growth and reaction characteristics within cell-immobilizing particles were investigated using the ethanol production system by Zymomonas mobilis to clarify the effect of mass-transfer resistance caused by cell growth on the overall reactivity of the immobilizing particles. For the immobilization of growing cells, the cell concentration at each position in the immobilizing particle increases monotonously with time, and after a certain period of cultivation the cell distribution and overall production rate reached steady state. However, the non-monotonous change with a peak of the production rate was observed with the elapse of time in the transient state. A novel method is proposed to restrict the overgrowth and consequently to enhance overall productivity in the steady state of the immobilized-cell biocatalyst, i.e., co-immobilization of microporous solid particles with growing cells. Also, a method to estimate the extent of the influence of co-immobilization of microporous particles was demonstrated, using a mathematical model.

Investigation of production of dextran and dextransucrase byLeuconostoc mesenteroides immobilized within porous stainless steel

Cells of Leuconostoc mesenteroides were immobilized within porus, stainless-steel (SS) supports and used for dextransucrase (DS) and dextran production. The pore size of the support significantly affected the dextran yields, which were greatest with average pore sizes of 2-5 microm. All immobilized-cell biocatalysts in porous stainless steel produced higher yields than free cells, with the exception of cells confined in submicrometer pores (0.5 microm). Coating supports of larger pore size (40 and 100 microm) with calcium alginate enhanced the cell-loading capacity of the supports and increased dextran and fructose yields in the cell-free broth. Controlled, fed-batch, DS production (activation), as a step preliminary to dextran production, significantly improved the subsequent dextran and fructose yields and shortened the time required to attain the maximum such yields. Scanning electron microscopy (SEM) of immobilized L. mesenteroides in stainless steel shows an irregular pattern of the microorganism inside the pores of the solid supports. Coating the porous solid supports with a cell-free calcium alginate layer led to an increase in the cell density inside the support. Cell growth inside the coated, porous stainless steel had no distinct growth form.

Immobilized cell biocatalyst activation and pseudo‐steady‐state behavior: Model and experiment

An intrinsic, unstructured model has been utilized to describethe startup dynamics of a continuous Caalginate-immobilized Zymomonas mobilis (ATCC 10988) fermentation. This model predicts, at least qualitatively, transients in the fermenter effluent glucose, ethanol, and biomass concentrations as well as radial gradients in immobilized-cell concentration and activity within the gelbiocatalysts. Predicted intrabiocatalyst gradients in immobilized-cell specific growth rate were used to calculate the corresponding gradients in intracellular RNA level based on a reported linear relationship between the two. Mathematical simulations of immobilized biomass concentration profiles and RNA content were verified using a novel, scanning microfluorimetry technique.

Product Inhibition Influence on Immobilized Cell Biocatalyst Performance

AbstractThe potential steady‐state influence of product inhibition on the performance of immobilized cell cultures is thoroughly explored with the aid of a simple unstructured reaction‐diffusion model. Production inhibition was assumed to diminish the specific growth rate directly. The range of product sensitivity explored corresponds directly with the observed range of ethanol sensitivity of Clostridium thermocellum, Clostridium ace‐tobutylicum, Saccharomyces uvarum, Zymomonas mobilis, and Saccharomyces cerevisiae. The consequent influence on the immobilized cell growth rate, thickness of steady‐state viable cell layer, formation of growth‐associated product, and global performance of the immobilized cell particle is explored by simulated variation of both the external bulk solution product level and the cell intrinsic sensitivity to the inhibitory product. Thus, both the thickness of the steady‐state viable cell layer and the consequent biomass loading of the bead are predicted variables of the model; these are variables due to the adaptive growth and biomass redistribution which occur in porous supports. The calculated influences are appreciable and even dramatic under some circumstances; the implications for packed‐bed reactor operations for ethanol and solvent production are discussed.These model calculations indicate that product inhibition can very significantly influence the performance of immobilized cell particles. Immobilization alone necessarily increases the impact of product inhibition on the culture since intraparticle product levels must be higher that external solution conditions. The presence of product inhibition lowers the specific growth rate of the culture pointwise in the carrier, thus diminishing the particle conversion of substrate to product and the total productivity and specific activity of the biocatalyst. Counterintuitively, perhaps, increased sensitivity to product inhibition results at first in an increased viable biomass loading of each carrier particle; then at still higher sensitivities a decreased loading and eventual extinction of viable cells is predicted. The application of these calculations for packed‐bed reactor design is suggested and constitutes a portion of our ongoing studies.

Scanning Microfluorimetry of Ca-Alginate Immobilized Zymomonas Mobilis

Scanning microfluorimetric techniques offer a means for in situ investigation of spatial heterogeneities in biomass density and cell composition in macroscopic, aggregate-cell systems such as immobilized-cell biocatalysts, biofilms, mold pellets and tumors. Much of the analytical power of flow cytometry, with the important addition of spatial resolution, can be brought to bear on the study of these systems. The utility of these techniques is demonstrated for the experimental determination of spatial gradients in biomass density and bio-catalytic activity in Ca-alginate beads containing an entrapped, living bacterium, Zymomonas mobilis ATCC 10988. Using cell RNA content as an indicator of cell growth rate, scanning microfluorimetry data indicates that immobilized cells in a single gel bead can exhibit the fullest possible variation in specific growth rates, from maximal to resting. Thus, clear gradients in biological growth rate within such biocatalysts are quantifiable, with implications for any fully or partially growth associated products.