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2.6 x 104

5.2 x 103

1.6 x 103

Note: Adding 20 mL of column A to 80 mL polymer solution leads to a fifth part of cells in column B. Assuming a survival rate for all batches of 31% leads to the number of colonies/mL LentiKat (column C).

Note: Adding 20 mL of column A to 80 mL polymer solution leads to a fifth part of cells in column B. Assuming a survival rate for all batches of 31% leads to the number of colonies/mL LentiKat (column C).

b. Six 100-mL bottles containing 80 g of LentiKat®Liquid were heated in a waterbath until the polymer solution was completely clear and put in a drying oven at 60°C. Additionally, 1 L of LentiKat®Stabilizer was prepared.

c. Cells from the preparatory culture shortly before the end of the exponential period of growth were taken and the number of colony forming units (CFU) was counted using a hemacytometer. Twenty milliliters were added for each batch. Batch 1 was undiluted, batches 2 through 6 were diluted by media as shown in Table 1. The resulting number of CFU is also given Table 1 and the survival rate is taken into account.

d. The survival rate is determined by counting the number of stained colonies from at least 20 LentiKats from batch 6 and generating the average value. Then the theoretical number of colonies for a LentiKat of this batch was calculated. From each batch, one Petri dish was printed with 413 LentiKats by the LentiKat®Printer so that 2.55 g of the polymer cell suspension is on each dish. At a density of 1 g/mL, and the assumption that the catalysts re-swell to their original size, 1, LentiKat has the volume of 6.17 |L and a mass of 6.17 |g, respectively.

e. The printed Petri dishes were dried under an exhaust hood with an extra fan installed 50 cm above the dishes. The LentiKats were dried to a residual mass of 28% (in this case when the droplets on one Petri dish weigh 0.714 g from the initial 2.55 g). This took about 40 min but this time is strongly dependent on humidity, temperature, air draught, and the size of the droplets. Times can vary from 20 to 80 min. The previously prepared LentiKat®Stabilizer is then poured on the LentiKats for re-swelling and for sloughing the gel particles. The LentiKats were transferred into an anaerobic workbench and put into media.

f. The theoretical number of colonies per LentiKat for batch 6 is, in this case, 32 (the observed average was only 10). This is equivalent to a survival rate of 31% which is assumed for the other batches as well because they were all treated in the same way (e.g., contact time to the polymer, drying time, contact time to oxygen, temperatures).

g. All batches were incubated anaerobically and stirred at 30°C at a start pH of 5.0 and a glucose concentration of 150 g/L. The media was changed twice a day. The

Fig. 7. Six batches with different initial biomass content (see Table 1, column C). (A) Specific activity of the batches after reaching a steady-state of activity. (B) Stained LentiKats from the different batch.

specific activity of the 6 batches was determined after 1, 4, and 6 d and found stable after 4 d. For the activity test each batch was incubated for 2 h and washed threefold with anaerobic media at constant temperature (30°C) before starting the real activity test in a 20-fold excess of media. The ethanol concentration is measured at the beginning and then every hour until the supernatant of the batches becomes turbid h. In this case the optimal initial biomass is 1.2 x 106 (see Fig. 7), which is obviously lower than the rough rule of thumb given before.

i. To produce highly active LentiKats with Zymomonas mobilis the initial biomass content should be near to this optimum. Keep in mind that the survival rate within immobilization can vary strongly with the state of growth and should be controlled for each immobilization batch.

9. Byconversion ofrawglycerol to 1,3-propanediol by Clostridium butyricum immobilized in LentiKats (4). A growing interest in 1,3-propanediol as a new commodity chemical has been observed over revent years. 1,3-Propanediol has a great potential as a new compound for the production of polymers with excellent chemical and mechanical properties.

There exists an interesting alternative to synthesis of 1,3-propanediol based on crude oil because various bacterial strains among the Clostridium butyricum are able to convert glycerol to 1,3-propanediol under oxygen exclusion with good yield.

By entrapment of C. butyricum in LentiKats the efficiency of biotechnical 1,3-propanediol production can be improved. This hydrogel entrapment offers the opportunity to run the process under nonsterile conditions over several weeks without any risk of contaminating microorganisms. Thus, entrapment into the gel lenses leads to a simple but stable process.

With immobilized cells of C. butyricum, a productivity of more than 30 g 1,3-propanediol per liter per hour was achieved in continuous fermentation thatt is 5-to 10-fold higher as compared with conventional fermentation. Raw glycerols from biodiesel production wre utilized for bioconversion to 1,3-propanediol without any loss in activity.

10. Significant reduction of energy consumption for sewage treatment by using LentiKat-encapsulated nitrifying bacteria (5,6). Bacteria from an external fermentation were employed in entrapped form to specifically increase the nitrification rate in nitrifying waste-water treatment plants. For maximum biological and mechanical stability, LentiKats were chosen as immobilization support. Wastewater from a municipal wastewater treatment plant was used directly for the lab-scale setup and the parameters ammonia, nitrate, and chemical oxygen demand (COD) were measured regularly. The setup was run with different parameters over a period of 650 d. A specific volumetric nitrate production rate of approx 25-30 mg/(Lh) was achieved at maximum nitrification. The residence time was between 30 and 60 min, which is 10 times shorter as compared with conventional methods. For real wastewater treatment plants this means a significant reduction in size for the nitrification reactors. The COD consumption showed values between 10 and 50% because of specific nitrification. The remaining COD is available for the subsequent denitrification step. The LentiKats showed no deterioration over the complete time span.

11. Bioethanolproduction from nonsterile molasses by yeasts. Bioethanol is one of the most suitable energy carriers to reach the goal of the European Union to increase the biofuel rate to 5.75% in 2010 for transport purposes.

In a three-step pilot scale process with yeasts immobilized in LentiKats, a productivity of 18 kgEtOH/(m3 h) could be obtained from nonsterile molasses without pH adjustment. The productivity of industrial production (batch, free cells) is in the range of 1.3-1.5 kgEtOH/( m3h). Based on these results a conventional process concept was compared with of a process concept with yeasts immobilized in LentiKats. Only three fermenters with 60 m3 each were needed instead of six fermenters each with 200 m3 to produce 2500 L/h bioethanol.

12. Production of(R)-cyanohydrins by using entrapped (R)-oxynitrilase in LentiKats (7). The (R)-oxynitrilase (hydroxynitrilase lyase, E.C. 4.1.2.10), which catalyzes the reversible condensation of hydrogen cyanide with aldehydes and ketones, is a useful and promising enzyme for biotransformations. The resulting optically active cyanohydrins are versatile synthetic intermediates to prepare several important classes of chiral compounds: a-hydroxy acids and their esters, P-amino alcohols, P-hydroxy-a-amino acids, a-hydroxy aldehydes, and a-hydroxy ketones. These compounds are important precursors for pharmaceuticals and agrochemicals.

Because costs for the enzyme and the efforts for recovering the soluble bio-catalyst from the reaction mixture are high, an industrial use is not economic yet. By entrapping the enzyme a repeated use is possible and thus efficiency of the process is raised. Of course the applied immobilization technique has major impact on technical feasibility: it has to be effective and cheap at the same time.

The activity of (R)-oxynitrilase was assayed by synthesis of (R)-mandelonitrile.

The reaction was performed in a weak acidic buffer solution (pH 3.8) at a low temperature (20°C) to diminish the competing nonenzymatic formation of a race-mic mixture. Excellent enantiomeric excess (>99% ee, determined by HPLC analysis) was obtained.

For industrial use of enzymes the long-term stability is a crucial economical criterion. Hence, the (R)-oxynitrilase was entrapped in hydrogels to study the efficiency and the long-term stability of this system. The entrapment of (R)-oxynitrilase in hydrogels was based on a very simple two-step procedure. First, the molecular weight of (R)-oxynitrilase (60,000 g/mol) was increased by cross-linking and then the cross-linked enzyme was entrapped in LentiKats. The activity of the cross-linked (R)-oxynitrilase was 65% of its native activity; after entrapping, 89% of this activity remained. The entrapped (R)-oxynitrilase was used repeatedly and the activity was stable for at least 25 batches.

References

1. Jekel, M., Buhr, A., Willke, T., and Vorlop, K.-D. (1998) Immobilization of biocatalysts in LentiKats®. Chem. Eng. Technol. 21(3), 275-278.

2. Czichocki, G., Dautzenberg, H., Capan, E., and Vorlop, K.-D. (2001) New and effective entrapment of polyelectrolyte-enzyme-complexes in LentiKats. Biotechnol. Lett. 23(16),1303-1307.

3. Jahnz, U. and Wittlich, P. (2004) GeniaLab BioTechnologie Produkte und Dienstleistungen GmbH. Available at http://www.genialab.com/download/tt-english.pdf. Accessed 03/24/2004.

4. Wittlich, P., Schlieker, M., Lutz, J., Reimann, C., Willke, T., and Vorlop, K.-D. (1999) Bioconversion of glycerol to 1,3-propanediol by LentiKatsTM. SchrR. Nachwachsende Rohstoffe 14, 524-532.

5. Sievers, M., Schäfer, S., Jahnz, U., Schlieker, M., and Vorlop, K.-D. (2002) Significant reduction of energy consumption for sewage treatment by using LentiKat® encapsulated nitrifying bacteria. Landbauforsch Volk SH 241, 81-86,

6. Sievers, M., Vorlop, K.-D., Hahne, J., Schlieker, M., and Schäfer, S. (2003) Advanced nitrogen elimination by encapsulated nitrifiers. Water Sci. Technol. 48(8), 19-26.

7. Gröger, H., Capan, E., Barthuber, A., and Vorlop, K.-D. (2001) Asymmetric synthesis of an (R)-cyanohydin using enzymes entrapped in lens-shaped gels. Org. Lett. 3, 1969-1972.

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