Enzyme Immobilization

2. Stirring controller

3. Stirrer

4. Impeller shaft

5. Air pump

7. Airdiffuser

8. Cryo-thermostatlc bath

9. Internal heat exchanger

10. Dissolved oxygen and temperature sensor

Fig. 2. Stirred tank reactor.

2. Materials 2.1. Reactors

1. Stirred tank reactor. A common cylinder-shaped vessel with a working volume of 5 L (total volume 7 L) is proposed. The reactor design allows a homogeneous distribution of liquid, avoiding dead volumes and decreasing the hydrodynamic stress. The system incorporates a stirring controller that allows a stirring rate between 0 and 500 rpm. Appropriate agitation is achieved with a set of stainless steel cross-blades. A thermostatic bath is connected to an internal heat exchanger in order to achieve the optimum temperature for the cells. An air pump, fitted to an amicrobic filter, performs the aeration for the reactor through a porous diffuser located underneath. The gas leaves through another air filter. This system is completed with a dissolved oxygen sensor and the reactor is filled with PUF cubes (20-25 g). The reactor is represented in Fig. 2.

2. Packed column reactor. A PVC tube (0.89-m height) with a total volume of 2.38 L is proposed. The working packed volume is 2 L (20 g of PUF cubes). Three sample ports are located along the column for the removal of solid units, and two

Enzyme Immobilization Carrier
Fig. 3. Packed column reactor.

ports (input and output) for the sampling of liquid. The column is aerated from the base with an air diffuser that is connected to an air filter (0.22 |im pore size). The column is fed from the upper part with a peristaltic pump and the packed column is continuously submerged in the feeding liquid in order to avoid the formation of preferential routes and to obtain greater homogenization. The pump is linked with a nutrient tank (4 L), which contains an appropriate liquid medium for cell growth and is continuously aerated with an air compressor. The liquid effluent from the column feeds the nutrient tank. The column and the nutrient tank have a water jacket and are maintained at the optimum temperature. The system is represented in Fig. 3.

2.2. Preparation of Inoculum

Prior to the immobilization it is necessary to obtain a given quantity of free submerged cells to feed the reactors. Such an inoculum consists of a suitable fermentative medium with a selected strain of the cell in its exponential growth phase. This material provides the source of cells for the adsorption onto the foam. The appropriate literature for every microorganism provides particular protocols for the culture propagation, normally consisting of various scale-up steps. It is very important to point out that the activity of this culture is a decisive factor for the success of the subsequent immobilization.

2.3. Carrier Units and Sterilization

Cubic foam pieces (1-cm length) of commercial polyurethane are used. These units have a porosity of around 97% and an apparent density of 0.02 g/mL. The average pore size of the units is 400 ^m (see Fig. 1).

Polyurethanes can be synthesized by the reactions of an alcohol with two or more hydroxyl groups and of isocyanates with more than one isocyanate group. This type of reaction is known as an addition polymerization. A combination of hydrogen bonding and the nature of the R groups gives rise to the range of properties observed for polyurethanes. A foam is formed when CO2 gas becomes trapped within the polymer during the isocyanate reaction with water.

Both types of reactors are made from autoclavable materials and so they can be sterilized (120°C, 15 psi, 20 min) with the cubic foam units inside.

2.4. Analytical Methods

Different types of analysis have to be performed on the liquid medium and the carrier in order to follow the immobilization process.

1. Submerged biomass concentration in the liquid medium (viable and nonviable cells) is determined by optical microscopy using statistical re-count with a Neubauer chamber.

2. Substrate or product concentrations in the liquid medium must be analyzed with the aim of controlling the fermentation process.

3. Immobilized biomass concentration is measured using the following technique (8): a unit of carrier is removed from the reactor and squeezed lightly in order to remove the interstitial liquid. The carrier is then submerged in an Erlenmeyer flask containing 25 mL of a buffer solution with the optimum pH for the immobilized cell. The flask is placed in an ultrasonic bath at room temperature for 15 min. These conditions lead to the total desorption of adhered cells. In the final stage, the Neubauer chamber re-count method for the submerged cells is carried out on the liquid phase. The carrier is subsequently removed from the flask and dried in an oven at 80°C for 24 h. It is then possible to calculate the number of immobilized cell/mg of dry carrier . This technique must be validated prior to use by developing experiments concerned with cellular resistance to ultrasonic treatment.

4. Interstitial biomass concentration is calculated by removing the liquid inside the unit of carrier by light squeezing and determining the biomass occluded by means of optical microscopy using statistical re-count with a Neubauer chamber. As indicated above, this is an important reference to evaluate the success of the immobilization procedure.

3. Methods

The methodology of immobilization will be described for two different reactor configurations. The two systems described are the most commonly used in laboratory and industrial processes developed with the participation of fixed cells: stirred tank reactor working in a discontinuous regime (by cycles) and packed column working in continuous operation mode.

3.1. Discontinuous Stirred Tank Reactor

The general procedure involves carrying out consecutive fermentation cycles in a discontinuous operation mode, with the free microorganisms suspended in the liquid medium in the presence of the solid carrier (PUF cubes). Throughout the different cycles, the biomass gradually adheres to the carrier until a point of maximum adsorption capacity (biofilm saturation) is achieved. The experimental work is outlined in the following steps:

1. The 7-L stirred tank described in Subheading 2.1., item 1 is charged with 20 to 25 g of cubic units of PUF and the system is sterilized by wet heat in an autoclave.

2. 5 L of inoculum (growth medium with a large population of cells in exponential growth phase) is introduced into the reactor.

3. Optimum conditions for the growth of submerged cells are implemented in the reactor (e.g, aeration, stirring rate, temperature). The agitation causes the foam units to scatter throughout the liquid volume because of the low density of the foam. At this point, it is necessary to remember the importance of establishing a soft stirring rate, which guarantees appropriate levels of homogenization and oxygen transfer to the cell while avoiding the undesirable shear stress effects due to collisions between the units.

4. In the first cycle, the population of submerged cells will complete the consumption of the substrate and generate the product of the main biological reaction. The state of such cultures is followed by analysis of submerged biomass and evaluation of substrate and product concentrations.

5. When the cycle is complete, a sample of polyurethane foam (2 or 3 units) is removed from the reactor. A counting protocol for total immobilized biomass and interstitial biomass is then carried out. The next stage involves refreshing the liquid medium: a percentage of the total liquid volume is removed (approx 50%) and the reactor is then re-filled with the same volume of fresh growth medium (substrate). A new discontinuous cycle then starts.

6. Several discontinuous cycles are carried out (see scheme in Fig. 4). At the end of each cycle a sample of solid carrier is removed and analyzed. The concentration of immobilized cells progressively increases up to the maximum adsorption capacity. Normally, four to six cycles are sufficient (300-500 h). The immobilized cell concentration follows the trend shown in Fig. 5, which is explained in the Note 1.

7. Once the final saturation point has been reached, the carrier particles are ready to be used for fermentation purposes in the reactor or to be removed and used in another reactor.

3.2. Continuous Flow Through a Packed Column

This procedure involves circulating the culture medium through the aerated submerged column (from the top to the bottom), thus allowing the progressive fixation of the cells to the PUF cubes. The culture medium then flows back to the nutrient tank for a continuously recycling flow. The system can be summarized as follows:

1. The 2-L packed column described in Subheading 2.1., item 2 is filled with 20 g of cubic PUF units and the system is sterilized by wet heat in an autoclave.

Enzyme Immobilization
Fig. 4. Scheme showing discontinuous cycles carried out with the submerged culture in the stirred tank: Concentration of substrate vs time.
Enzyme Immobilization
Fig. 5. Profile for immobilized biomass concentration (in millions of cell/mg of carrier vs time) in PUF cubes submerged in a discontinuous stirred tank.

2. The reactor is cooled and connected to the rest of the equipment. The 4 L of inoculum in the nutrient tank is allowed to begin to fill up the column from the top. An external pipe keeps the column continuously submerged and maintains a constant level of liquid. The effluent exiting from the bottom of the column returns to the nutrient tank.

3. A wide range of residence times (HRT) can be established in the reactor but, below a critical flow rate, this factor has little influence on the immobilization rate and the total adsorption capacity. A time of 1 h is recommended.

4. It is necessary to analyze the submerged biomass, substrate, and product concentrations in the input and output liquid on a daily basis. Furthermore, three samples of PUF units (at different column ports) must be removed in order to estimate the immobilization degree of the carrier and the interstitial biomass. The nutrient tank must also be refreshed with new growth medium when the substrate has been exhausted.

5. A massive immobilization of cells will be registered almost immediately and will reach saturation on the carrier (steady state) in 200 to 300 h.

6. Once the final saturation point has been reached, the carrier particles are ready to be used for fermentation purposes in the reactor or to be removed and used in another reactor.

4. Notes

1. The trend followed by immobilized biomass, based on abundant experimental data, is shown in Fig. 5. This behavior can be explained in terms of the internal structure of PUF. After an initial stage of approx 100 h without any appreciable immobilization, a sudden increase in adhered biomass is observed (within the next 150-200 h). The maximum colonization of PUF is then reached. From this point on, no further adsorption in registered. This trend is directly related to the hydrodynamic behavior of the PUF submerged in the liquid phase. During the first hours of operation (discontinuous or continuous) the PUF cubes remain dry, but as the process continues they gradually become completely wet by capillary action. After that point, the adsorption begins at a high rate because of the highly porous structure of this carrier, which facilitates the total exposure of the surface and accesibility to the cells (8).

2. As indicated in the introduction, the stirring rate plays a very important role in determining the maximum adsorption capacity. Studies in the literature show that an increase in the stirring rate causes a decrease in the immobilization. As an example, the immobilization of Acetobacter aceti on PUF reaches 6 million cell/ mg of carrier when the stirring rate is 200 rpm but increases to 13 million when the agitation rate is decreased to 125 rpm (13).


1. Moonmangmee, S., Kawabata, K., Tanaka, S., Toyama, H., Adachi, O., and Matsushita, K. (200) A novel polysaccharide involved in the pellicle formation of Acetobacter aceti. J. Biosci. Bioeng. 93(2), 192-200.

2. Manohar, S., Kim, C. K., and Karegoudar, T. B. (2001) Enhanced degradation of naphthalene by immobilization of Pseudomonas sp. strain NGK1 in polyurethane foam. Appl. Microbiol. Biotechnol. 55(3), 311-316.

3. Moe, W. M. and Irvine, R. L. (2001) Polyurethane foam based biofilter media for toluene removal. Water Sci. Technol. 43(11), 35-42.

4. Hori, H., Yamashita, S., Ishii, M., Tanji, Y., and Unno, H. (2001) Isolation, characterization and application to off-gas treatment of toluene-degrading bacteria. J. Chem. Eng. 34(9), 1120-1126.

5. Moe, W. M. and Irvine, R. L. (2001) Effect of nitrogen limitation on perfomance of toluene degrading biofilters. Water Res. 35(6), 1407-1414.

6. Yang, C., Suidan, M. T., Zhu, X., and Kim, B. J. (2003) Comparison of a single-layer and multi-layer rotating drum biofilters for VOC removal. Environ. Prog. 22(2), 87-94.

7. Burgess, J. E., Parsons, S. A., and Stuetz, R. M. (2001) Developments in odour control and waste gas treatment biotechnology: a review. Biotechnol. Adv. 19, 35-63.

8. de Ory, I., Romero, L. E., and Cantero, D. (2004) Optimization of immobilization conditions for vinegar production. Siran, wood chips and polyurethane foam as carriers for Acetobacter aceti. Process Biochem. 39, 547-555.

9. Holubar, P., Plas, C., Weiss, B., Sasshofer, S., and Braun, R. (1994) Hydrocarbon removal with a polyurethane foam bioreactor. Biologische Abgasreinigung. 1104, 505-s510.

10. Armentia, H. and Webb, C. (1992) Ferrous sulfate oxidation using ThiobaciUus ferrooxidans cells immobilised in polyurethane foam support particles. Appl. Microbiol. Biotechnol. 36, 697-700.

11. Mori, A. (1985) Production of vinegar by immobilized cells. Process Biochem. 20(3), 67-74.

12. Van Loosdrecht, M. C. M., Eikelboom, D., Gjaltema, A., Mulder, A., Tijhuis, L., and Heijnen, J. J. (1995) Biofilm structures. Wat. Sci. Tech. 32(8), 35-43.

13. de Ory, I., Romero, L. E., and Cantero, D. (2000) Influence of shear stress on immobilization of acetic acid bacteria on polyurethane foam carriers. Mededelingen-Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen. Universiteit Gent. 65(3a), 235-241.

14. O'Reilly, A. M. and Scott, J. A. (1995) Defined coimmobilization of mixed microorganism cultures. Enzyme Microb. Technol. 17, 636-646.

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