Fermentation processes immobilized-cell

Continuous heterogeneous catalysis by fixed microbial cells represents a new approach to established fermentation processes. Immobilization of isolated (and purified) enzymes and microbial cells mediating simple, monoenzyme reactions has already been reduced to industrial practice. However, the development of immobilized cell systems to carry out complex fermentation processes—characterized by multiple reactions cind complete reaction pathways involving coenzymes—is still in its infancy. Drawing upon our rather concerted effort in this area over the past several years, we are appraising the prospects and problems of such a technological advancement in this brief communication. 

Genisheva Z., Mota A., Mussatto S.I., Oliveira J.M. and Teixeira J.A. Integrated continuous winemaking process involving sequential alcoholic and malolactic fermentations with immobilized cells. Process Biochemistry 49 (1) (2014) 1-9. 

In plant cell cultures, microencapsulation, by mimicking cell natural environment, improves efficiency in production of different metabolites used for medical, pharmacoIogicaL and cosmetic purposes. Human tissues are turned into bioartiflcial organs by encapsulation in natural polymers and transplanted to control hormone-deficient diseases such as diabetes and severe cases of hepatic failure. In continuous fermentation processes, immobilization is used to increase cell density, productivity and to avoid washout of the biological catalysts from the reactor. This has already been applied in ethanol and solvent production, sugar conversion, or wastewater treatment. 

The production of SA was studied with entrapped and adsorbed Actinobacillus succinogenes on varied supports (e.g., diatomite or zeolite, approx. 12.6 mg/g support) [136], Higher productivities in SA were achieved with A. succinogenes entrapped in agar beads. After 5 cycles (72 h) repeated batch fermentations with immobilized cells in agar, the production of SA was 107 g/L. These approaches reduced significantly the fermentation time and improved the process yield, productivity, and final concentration in SA [136]. 

Experimental fermentation processes employing cell immobilization have been applied to propionic acid production by the propionibacteria. The objectives are to create a stable high-... 

Although the use of fermentation to produce ethanol is an ancient technology, more efficient immobilized-cell, continuous processes have been conceived, and Japan has estabhshed the first demonstration-scale plant. 

The immobilization of microbial cells under conditions where an activity or set of enzymic activities remains intact, but the normal metabolic processes cease, represents a novel technique for enzyme immobilization. Moreover, immobilized cells might enable the standard fermentation methods to be replaced by immobilized -cell -based continuous processes. 

Many alternative fermenters have been proposed and tested. These fermenters were designed to improve either the disadvantages of the stirred tank fermenter-high power consumption and shear damage, or to meet a specific requirement of a certain fermentation process, such as better aeration, effective heat removal, cell separation or retention, immobilization of cells, the reduction of equipment and operating costs for inexpensive bulk products, and unusually large designs. 

For bioprocessing purposes, increase in the stability of biocatalysts is quite often achieved by immobilization of cells or enzymes [57-60], This technology is an attractive alternative to the use of expensive free enzymes and cofactors, and can coordinate multistep enzymatic processes into a single operation. Furthermore, fermentative biosynthesis of cephamycin C using immobilized cells of S. clavuli-gerus NRRL 3585 was accomplished by Freeman and Aharonowitz [61], Jensen et al. [62] reported on the immobilization of P-lactam synthesizing enzymes from the same wild-type culture. None of these early studies used penicillin substrates other than the normal intermediate penicillin N, such as penicillin G. 

Corn steep liquor (CSL), a byproduct of the com wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 hr1. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(Lh), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation. 

Optimizing the ABE fermentation process has long been the aspiration of more than a century of research. Conventionally, the profitability of fermentation is influenced by the type and concentration of substrate, dilution rate, pH, culture medium, and product recovery. Even using cell recycle, cell immobilization, or extractive fermentation to increase cell density and productivity, the yield of the combined ABE production never exceeded 0.44 g/g (13-15). 

A bioreactor is a vessel in which biochemical transformation of reactants occurs by the action of biological agents such as organisms or in vitro cellular components such as enzymes. This type of reactor is widely used in food and fermentation industries, in waste treatment, and in many biomedical facilities. There are two broad categories of bioreactors fermentation and enzyme (cell-free) reactors. Depending on the process requirements (aerobic, anaerobic, solid state, immobilized), numerous subdivisions of this classification are possible (Moo-Young, 1986). 

Yadav, B. S., Rani, U., Dhamija, S. S., Nigam, R, and Singh, D. (1996), Process optimization for continuous ethanol fermentation by alginate-immobilized cells of Saccharomyces cerevisiae HAU-1, J. Basic Microbiol., 36, 205-210. 

Separation of lactic and propionic acids. The lactose fraction in the sweet whey permeate from cheese whey ultrafiltration can be fermented to produce lactic acid. In conjunction with the fermentation step, inorganic membranes have been tested in a continuous process to separate the lactic acid. This approach improves the productivity and reduces energy consumption compared to a conventional fermentation process. In addition, it produces a cell-free product. In a conventional process, some cells, although immobilized, are often detached and released to the product Zirconia membranes with a MWCX) of 20,000 daltons were operated at 42 C and a crossflow velocity of 2-5 m/s for this purpose [Boyaval et al., 1987]. The resulting permeate flux is 12-16 L/hr-m. To... 

Glucose and lactate were simultaneously monitored by Wei Min et al. [191] during the fermentation of Lactococcus lactis. They made use of a microdialysis probe connected to a dual flow-through cell in which two amperometric biosensors were located. The biosensors were based on the co-immobilization of the respective oxidase enzymes together with HRP in a carbon paste matrix. Both analytes were monitored on-hne for about 14 h in a very complex aqueous two-phase fermentation process, and the results were in good agreement with independent ofiF-line HPLC measurements. 

Also, the cephalosporin-transforming activity of D-amino acid oxidase isolated from yeast was identified in a similar manner. The thermometric signal was proportional to the number of cells as well as the amount of D-amino acid oxidase immobilized in the ET microcolumn. The ET was also coupled to a thermometric ELISA procedure (TELISA) for the determination of hormones, antibodies and other biomolecules generated during the fermentation process... 

Three-phase fluidized beds can be used as bioreactors for aerobic biochemical processes, including both fermentation processes and wastewater treatment. The gas phase is air, required for biological growth, while the solid particles provide immobilized surfaces on or in which cell growth can occur. The aqueous liquid phase provides the culture medium needed for the growth and maintenance of the cells. Air may be introduced separately from the liquid, or be premixed with the aqueous medium. The liquid medium may exhibit non-Newtonian rheology. A disadvantage of three-phase... 

The first commercial production of L-aspartic acid was started in 1973 by the Tanaba Seiyaku Company, Japan. The process uses aspartase contained in whole microorganisms and involves the immobilization of E. coli on polyacrylamide gel or carrageenan. The immobilized cells are then subjected to treatment in order to increase cell permeability. The substrate, fumaric acid, is dissolved in a 25 % ammonia solution and the resulting ammonium fumarate is then passed through the reactor containing the immobilized E. coli. The reaction is exothermic and the reactor has to be designed to remove the heat produced. The conversion of fumaric acid to aspartic acid is more economical than the direct fermentation of sugars. The key to economical production of L-aspartic acid for expanded use is a cheaper and more abundant source of fumaric acid. 

These examples indicate the in situ applicability of enzyme electrodes however, numerous problems have still to be solved. At present, coupling of enzyme sensors for fermentation control in a bypass arrangement appears to be more favorable [412]. Following this concept, an invertase thermistor incorporating a sterilizable filter unit has been developed [413] for the monitoring of alcoholic fermentation by immobilized yeast cells. Another thermistor has been successfully used for on-line glucose measurement under real cultivation conditions of Cephalosporium acremonium [414]. Similar calorimetric devices are suitable for other fermentation processes and in environmental analysis. 

---