Marc Schlieker and Klaus-Dieter Vorlop
Immobilization by entrapment in hydrogel particles based on polyvinyl alcohol (PVA) shows many advantages in immobilizing living cells and cross-linked enzymes. The immobilization takes place at room temperature in aqueous environment. The outstanding advantages compared to other immobilization methods are described. Subsequently the different possibilities for forming these hydrogel particles are introduced and many practical tips are given for understanding important parameters like initial biomass content or the survival rate during the immobilization step.
The benefit of this immobilization technique is shown by some examples of successful immobilization of whole cells and enzymes in LentiKats®: bioconversion of raw glycerol to 1,3-propanediol, bioethanol production from nonsterile molasses, reduction of energy consumption for sewage treatment, and production of (^)-cyanohydrins by using entrapped (^)-oxynitrilase in LentiKats.
Key Words: Hydrogel; polyvinyl alcohol; PVA; LentiKats®; whole cell immobilization; growth inside porous gel structure; enzyme entrapment; nonsterile bioconversion; 1,3-propanediol; ethanol production; (^)-cyanohydrin; waste water treatment.
The outstanding advantages of immobilization in biotechnology impelled whole generations of scientists to search for suitable methods to use these benefits and to overcome the problems at the same time.
The main improvements are a significantly increased productivity, an easy separation that allows use in repeated or continuous processes, and a high process stability including a protection of labile cells. On the other hand, some drawbacks usually come along with an immobilization: diffusion limitation of the immobilized biocatalyst, deactivation during immobilization, and abrasion of the immobilization matrix during the use in bioreactors. Last but not least there are additional costs of immobilization.
Fig. 1. Sketch of a LentiKat.
Vv 200 -400 Mm
From many different immobilization methods, we chose entrapment in hydrogel particles. By preparing porous particles of natural or synthetic polymers around the biocatalyst, nearly every cell type or part of a cell can be immobilized. The most prominent examples are hydrogels of polysaccharide like carrageenan, pectate, and alginate. Hydrogel beads allow cell growth inside the gel and offer an almost optimal environment to perform bioconversions except for a low mechanical stability. High ratios of microorganisms (>30% [v/v]) or intense gas evolution can lead to bead disruption. In the case of calcium alginate beads the presence of chelators (e.g., phosphate buffer), may lead to deterioration. In addition to that the immobilization matrix is highly biodegradable.
To overcome the problems of these natural polymers and benefit from the great advantages at the same time one can switch to polyvinyl alcohol (PVA)-based matrices. PVA is a synthetic polymer that can be used to form hydrogels by the conventional freeze-thawing method and leads to particles with excellent mechanical properties.
A new method to produce lens-shaped hydrogels based on this material at very gentle conditions, room temperature, and short time has been developed (1).
A LentiKat (see Fig. 1) combines the advantages of small and large particles: minimized diffusion limitation caused by the thin structure is linked to easy separation because of the large diameter of the biocatalyst. It can be retained easily in the bioreactor (e.g., by a sieve).
In contrast to biopolymers PVA hydrogels are hardly biodegradable and show an excellent mechanical stability. A large elongation at break (350-450%) and even in long-time fermentations no significant abrasion could be observed.
Correctly stabilized LentiKats tolerate a maximum temperature from 50 to 55°C, and pH values between 3.1 and 8.5 were tested for several days or weeks without any signs of disintegration of LentiKats. The suitability for even sensitive biocatalysts is given by the gentle entrapment conditions—only a short time is needed at room temperature.
The inner pore structure of the matrix can be adapted by additives according to the needs of the cell or enzyme.
The principle of gelation is a partial drying of the biocatalyst-loaded polymer solution. The polymer solution (LentiKat®Liquid) is mixed with the biocatalyst, droplets have to be formed and dried to a certain water content (see Fig. 2). After a re-swelling step the LentiKats are ready to use.
1. LentiKat®Printer (GeniaLab, Braunschweig, Germany).
2. LentiKat®Liquid (GeniaLab; see Note 1).
3. LentiKat®Stabilizer (GeniaLab).
5. Sodium chloride (Merck, Darmstadt, Germany).
6. Carbol-Fuchsin solution (Merck).
3.1. Preparation of the Polymer-Biocatalyst Solution
The LentiKat®Liquid (see Notes 1 and 2) has to be melted by placing in a water-bath between 85 and 90°C. Use a magnetic stirrer for thorough mixing. Melt the polymer solution until the complete content of the bottle is a homogenous and clear liquid. The solution has to be cooled down to a temperature that is adequate for the biocatalyst directly before the immobilization process to avoid an undes-ired gelation process that will occur at lower temperatures. However, at a temperature between 25 and 30°C LentiKat®Liquid will be workable for several minutes.
After cooling, the biocatalyst solution is added and dispersed homogenously by mixing with a magnetic stirrer. In the case of immobilizing biomass wet matter (e.g., cells separated by centrifugation) the biomass has to be diluted by at least 50% with 1% sodium chloride solution before adding to LentiKat®Liquid.
3.2. Production of LentiKats
For production of the LentiKats a smooth plate is needed, preferably of polystyrene. For example, single-use Petri dishes give good results. Some materials (e.g., glass) are not suitable at all. Each plate has to be marked individually to be weighed and the tare noted (see Note 3).
Using a standard syringe with a cannula (~1.0 mm in diameter), droplets need to be formed and dripped neatly and rapidly onto the surface of the plate (see Note 4). The droplets should be approx 3 mm in diameter, weigh 5 mg, and be as uniform as possible. After finishing a complete plate the amount of LentiKat®Liquid on this plate can be determined by balancing the whole plate again.
3.2.2. Using a LentiKat® Printer
For reproducible experiments in shaker flasks or small fermenters uniform particles are needed (see Notes 5-8). The easiest way to produce small amounts of immobilized catalyst is to use a special tool: the LentiKat®Printer (see Fig. 3). In one step, more than 400 uniform particles are formed and deposed on a Petri-dish. For a detailed description see "tips and tricks" in ref. 3. Up to 50 g of LentiKats can be produced easily in lab-scale, even under sterile conditions.
A smaller printer head is available with only 120 pins for smaller batches and reduced losses of material. This type is recommended when entrapping enzymes or other valuable materials.
After calculating the mass of the droplets the gelation by partial drying has to be launched. Drying can be done by leaving the plate simply exposed to air but to accelerate the evaporation of water a ventilator or fan for aeration from above is useful. Maximum temperature during gelation should not rise above 35°C to avoid harm for the biocatalyst.
Drying of LentiKats is complete if 72% of the mass of LentiKat®Liquid that has been dripped on the plate has evaporated. For this reason it is advisable to control the weight of the plate with LentiKats regularly during gelation. The time needed for gelation strongly depends on the air draught and its temperature and humidity.
When gelation is complete the previously prepared LentiKat®Stabilizer is poured on the LentiKats for re-swelling and for sloughing the gel particles. LentiKats will reswell to about their original size by this treatment. After 3 to 8 min of contact with the stabilizer solution LentiKats are easily removed from the surface and put into a bottle containing a 10-fold surplus of LentiKat®Stabilizer. LentiKats have to be stirred for at least 2 h in stabilizer solution to give a good mechanical stability for the later application. The influence on LentiKat stability of any shortening in the times of operation given above have to be proved for each case.
After stabilizing is finished the supernatant has to be removed and replaced by the medium or solution with which you want to use your immobilized biocatalyst. If foaming occurs it may be necessary to remove and replace supernatant again after a certain time of stirring.
Some specific examples of the use of this system may be found in Notes 9-12.
For an industrial application using LentiKats a scale-up of the LentiKat production is necessary. We built up a pilot-plant for a continuous production in our institute (see Figs. 4 and 5). We chose a belt dryer using pretreated and dried air. A multinozzle system puts rows of 23 droplets on the belt.
After controlled drying in a 15-m tunnel the droplets are passed to a re-swelling area. A wiper takes the lenses off the belt and they fall into a stabilizing bath for hardening. Temperature and humidity are measured and controlled at different points in the drying tunnel and can be adjusted towards the desired water content of the droplets.
1. Commercialized LentiKat®Liquid is shipped sterile in a ready-to-use form. In the bottle are 80 g of the PVA-solution with various additives designed to mix with 20 mL of the biocatalyst solution resulting in 100 mL of LentiKats.
2. LentiKats are not only suitable for the immobilization of whole cells but also for enzymes (2).
3. These aspects should be taken into account before starting to immobilize:
a. How much LentiKats should be prepared? Can you use a syringe or do you need a LentiKat®Printer?
b. Are enough (sterile) Petri dishes and LentiKat®Stabilizer available?
c. Do you want to use the LentiKats directly after the production or do you want to store them? Do you have you enough media? Does storing harm the entrapped biocatalyst?
4. Microorganisms entrapped in LentiKats can be controlled microscopically. To enhance contrast of cells and hydrogel, staining may be useful: treatment with
1:100 diluted Carbol-Fuchsin solution (ZIEHL and NEELSEN) gave good results after staining and decolorising of 30 min each.
5. If you are working with your biocatalyst entrapped in LentiKats you probably do not have to be as attentive to sterile conditions. Contaminating cells will not be able to enter the hydrogel and replace the wanted biocatalyst which hence will be in great surplus compared to any contaminating cells.
6. Initial biomass of living cells. If you are doing entrapment of living cells that should form colonies inside the LentiKats after immobilization the initial biomass content will without any doubt strongly influence the maximum specific activity you will find after growing to a steady state.
• If initial biomass content is too high you will get shell-catalyst characterized by intensive biomass growth at the surface of LentiKats leading to very poor nutrient supply in the central part of the gel and bad specific activity (see Fig. 6B). If initial biomass content is too low the gel volume of LentiKats is not utilized sufficiently (see Fig. 6A). Hence, optimum for initial biomass content has to be determined for each application. As a rule of thumb, an initial biomass content of about 107 cells/mL of gel should be near optimum. This value was found for different bacterial strains. The cell number is more useful than weight of biomass since cells of different strains may strongly differ in their size, but one colony will have its origin in one cell not in a certain small portion of biomass.
• If biomass content of LentiKats is too high some rolling of LentiKats may occur after some prolonged time of incubation. Besides decreasing biomass content there may be some relief by enlarging thickness of LentiKats or enhancing hydro-gel stability (duration of stabilization or further drying if possible).
• If dead or resting cells are to be entrapped a biomass content of 10% (w/w) biomass wet matter is recommended. If intracellular enzymes in more or less perforated cells ought to be used cross-linking the cell contents with glutardialdehyde might be helpful to prevent leakage of the biocatalyst.
7. The survival rate of microorganisms during immobilization can be determined by immobilizing a very small number of cells (e.g., 104 CFU/mL) and count the emerging colonies after staining. Subject to the assupmtion that the density of the biocatalyst polymer suspension and the LentiKats are about 1 kg/L and the hydro-gel is re-swelling to its primal volume the theoretical number of colonies per LentiKat can be calculated and set to 100%.
8. The immobilization of the bacterium Zymomonas mobilis in LentiKats is described in detail in the following steps:
a. To determine the optimal initial biomass content for this bacterium in LentiKats six batches with different initial biomass content were produced and their activity measured after they had grown to a steady state.
Numbers of CFUs/mL Cell Suspension
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