A METHOD AND APPARATUS FOR CONTINUOUS PREPARATION OF BIOCATALYST PARTICLES

Abstract: Continuous process for preparation of biocatalyst particles where biocatalyst (1) is mixed with hydrocolloid gelling agents, e.g., alginate (2), in a mixing chamber (3), the blended mass is further conducted in a tube (4) to a rotating, perforated cylinder (5), where said cylinder (5) is placed in a reactor (6), and the cylinder (5) is rotated at a velocity causing the beads to be hurled directly down into the reactor liquid (11).

Description: A METHOD AND APPARATUS FOE CONTINUOUS PREPA ATION OF BIOCATALYST PARTICLES 5 The invention relates to a method and apparatus for contin¬ uous preparation of biocatalyst particles, where the particles have uniform size and spherical shape. .- The term biocatalyst in this connection refers to a biologi¬ cal system comprising microorganisms or cells, as well as parts thereof, e.g., enzymes. By biocatalyst particles is meant biocatalysts that are fixed (immobilized) by means of a macroscopic carrier, e.g., a hydrocolloid gel, enabling the 15 system to carry out metabolic processes. Industrial utilization of biocatalyst particles has been limited by the possibility of producing such particles. Various processes for producing these particles have been 20 described previously. The simplest method is described in German patent DE 2835875 and is based on dripping an alginate solution out of a tube having a small diameter. The disadvantage of this method is 25 that the size of the alginate beads (due to the high viscosity of the solution) is almost independent of the tube dimension and therefore cannot be varied in a simple way. A further development of the aforementioned procedure is to j - blow out the droplet with pressurized air. It is thereby possible to vary the size of the droplets. The problem is the high production costs required for this method. A third method is to subject the alginate solution to - oscillation, thus causing the droplets to "shoot" out of a thin tube. This method has ample capacity, but produces varying sizes of alginate beads. Industrial evaluation of this method has found it to be too costly. The fourth method is described in DE 3417899 C2 "Verfaren zur Herstellung perl δrminger Biokatalysatoren und zur Steigerung der Durchsatzkapazitat und Vorrictung z * u einer Durchfiirung. " Said patent describes a production method for spherical immobilized biocatalystε. In this method alginate droplets are hurled out of a perforated cylinder and caught by a Z rotating centrifuge basin filled with CaC^-soTution. Due to the rotation, the CaCl2 solution climbs up along the wall of the centrifige basin. The rotating, perforated cylinder is placed down in the centrifuge basin so that the alginate beads that are hurled out migrate horizontally until they hit - the CaCl2 solution. The method has a high capacity and the size of the alginate beeds may be varied. The disadvantage of this method is that it is mechanically complicated and difficult to use in aseptic processes. By aseptic process, in this connection, is meant a process that does not contain any bacteria other than the desired ones. The alginate beads produced will end up in the rotating centifuge basin, and from there must be transferred continuously, in some way or other, to a production reactor. The invention's contribution is to move the perforated, rotating cylinder into the actual production reactor, which may also be an open and non-aseptic vessel. The method is characterized in that said cylinder (5) is placed In a reactor (6), and approximately monodisperse and approximately spherical particles are produced by rotation of the cylinder (5) at a velocity which hurls the beads directly down into the reactor liquid (11), and that the size of the beads is a function of the cylinder's velocity of rotation, which normally lies between 2.5 and 6300 /ε^, preferably between 2.5 and 360 m/ε 2 . The hydrocolloid droplets that are hurled out are now picked up by the surface of the reactor, as shown on Figure 1. The need for complicated mechanical equipment such as a centrifuge is thus avoided. By producing the alginate beads in the production reactor, one also avoids the complicated step of transferring the hydrocolloid beads from ■*, the centrifuge to the reactor, and the system may be used for 5 aseptic processes. With the process according to the , invention, spherical particles of uniform size are produced, and the beads are metabolically active from the instant they hit the liquid surface in the reactor. It is not necessary for the beads to be placed in special liquids/mixtures for a ; , period of time in order to mature. Nor is it necessary to separate them from the CaCl liquid as described in the German patent DE 3417899. The size of the particle beads that are hurled out may be 15 regulated by means of the velocity of the rotating cylinder. With the method described in the invention, we obtain particles having approximately monodisperse spherical shape and a size deviation of up to 10$. 20 The invention will be further explained below, with reference to Figure 1 showing the rotary device for dispersing the beads (hereafter referred to as the droplet or bead hurler ) placed in a potential reactor having supply lines for a gelling composition and biocatalyst, and a mixing chamber. 25 It is an advantage in the process according to the invention that the biocatalyst (1), e.g., bacteria, be rapidly transferred from a culturing apparatus (not shown) over to the mixing chamber (3) in order to avoid reducing the production potential. In mixing chamber (3) a solution of hydrocolloid, e.g., alginate (2), is mixed with the bio¬ catalyst (1), to arrive at the desired concentration for the components. Then the mixture is conducted via a rigid tube (4) to the rotating, perforated cylinder (5) placed in the reactor (6). This rigid tube (4) is connected to a device (7) for regulating the velocity of rotation of tube (4) and cylinder (5). The reactor may also be equipped with a feed pipe (9) for supply of substrate and chemicals, a discharge means (10) for tapping biocatalyst particles, a stirring device (12), and an outlet (10) for removal of the product. The droplet hurler (5) comprises a hollow cylinder sealed at one end thereof. The diameter of the cylinder may vary according to desired capacity. The upper end of the cylinder is connected at the center thereof to a rigid, hollow tube (4). This tube has two functions. When the tube rotates, the cylinder (5) also rotates with the same velocity and the hydrocolloid solution can be sent through the tube and down to the cylinder. The rotating cylinder has holes (8) drilled through the cylinder wall, enabling the biocatalyst solution in the hollow cylinder to be hurled out. Holes (8) may be distributed around the entire cylinder wall, and the diameter, placement and number of holes may vary depending on the desired capacity. The landing radius for the droplets, which determines the minimum radius for the reactor, can be computed from the following formula: where r: radius- v: velocity of the droplets as they leave the hurler 5 . h: height of the holes above the liquid level g: acceleration of gravity The formula's validity is shown in Example 1. ,, An important feature of the invention is that the size of the alginate bead is related to the velocity of rotation of the droplet hurler. This relationship can be determined by beginning with a droplet that is dripped out of a tube. The volume of such a droplet is expressed by the following equation: 2ττr-p V = 0 * d' where V = volume of the droplet r = diameter of the tube p = surface tension of the liquid d = density of the liquid g = acceleration of gravity ø = correction factor depending on the shape of the tube end The variation in bead diameter for the gelled alginate droplets as a function of the velocity of rotation can be found by converting the equation. The droplet'ε volume is given by 4πE 3 /3, where E is the radius of the alginate droplet. Acceleration, in our case, is proportional to the velocity of rotation in the second power. The other factors entered in the relation can be combined into one simple correlation factor: A. E = A (rpm)' where R = the diameter of the gel bead A = correlation factor rpm = revolutions per min. The validity of the formula is shown in Example 2. The described bead hurler has a high capacity, rendering it suitable for industrial use. The capacity in Example 1 and Example 2 is measured at 0.200 - 0.255 1 alginate solution per hour and hole at velocity of revolution of 500 to 1000 rpm. For the capacity measurement a bead hurler aε described in Example 1 was used. If the bead hurler is produced with 100 holes, capacity will lie at about 20 1 alginate εolution per hour, and this is sufficient for most purposes. The formation of alginate beads from alginate solution requires a water εolution of calcium ionε (5-200 mM) in the reactor. Since anaerobic and facultative anaerobic bacteria, aε well aε a number of enzymeε εuch aε proteaεeε and lipaεeε, produce acidε such as, respectively, energy metabolism products and reaction products, calcium ions can be added to the reactor as a neutralizing agent (for example Ca(0H)2), either alone or in combination with other basic compounds. The addition of soluble calcium saltε is also possible. If, e.g. , milk, whey or other milk products are used in the reactor, the milk or milk productε themselves will contain calcium ionε, making it neceεsary only to add small extra amounts of calcium salts. The reactor in the procesε may be of any type whatsoever that may be used with immobilized biocatalyst particles. The reactor thus may be, for example, a mixing tank reactor. A 25 sugar solution, for example dairy effluent, whey, or ultrafiltered whey, is conducted continuously (9) into the reactor, and product εolution iε removed (13) at an approxim¬ ately equal rate. >- With a εimple device (10) consisting of two computer- controlled ball valves and an intermediate volume, alginate beads may be tapped from the reactor so that the biocatalyst concentration in the reactor is held constant. ->? Example 1 Correlation between estimated landing radius and experimen¬ tally measured radius The solution of Na-alginate ( 2% w/v Protanal LF 10/60) is hurled out of the rotating droplet hurler. The droplet hurler has an outer diameter of 4.6 cm and 24 holes having a diameter of 1.3 mm drilled into the inner cavity of the cylinder. This cavity is in contact with the hollow shaft on which the cylinder iε mounted. Through the shaft the cylinder is supplied continuously with alginate solution. TABLE 1 Estimated and measured values for landing radius at varying velocities of rotation for the droplet hurler. Velocity of rotation Landing radius (cm) rpm Theoretical Experimental 310 9.6 8.1 380 11.8 12.0 470 14.6 14.5 590 18.3 18.5 780 24.2 23.0 930 28.8 27.0 1150 35.7 30.5 Example 2 Estimated and measured values for alginate bead size as a function of velocity of rotation of the droplet hurler The droplet hurler used is described in Example 1. The size 2of the alginate beads was measured with the aid of Dynatech Diffusion Zone Eeader -vlk II. TABLE 2 The size of the alginate bead as a function of the velocity of rotation of the droplet hurler. 5 Radius rpm mm Eelative correlation factor 1000 1.11 100 900 1.11 90.9 ,„ 800 1.33 100 700 1.67 115 600 1.89 113 500 2.55 135 , 5 Example 3 Production of alginate immobilized lactic acid bacteria The homofermentative lactic acid bacterium Lactobacillus helveticus CNEZ 303 iε εtationarily cultivated in medium (0.5 0 1) at 40°C. After 20 hours the culture iε innoculated in εanitary or, if desired, sterilized cultivation equipment, e.g., the membrane reactor εhown in the parallel Norwegian application, ref. no. 28140. 5 A sugar solution in the form of ultrafiltered whey and nitrogen source is conducted into the system. To the whey- are added inorganic nutrientε aε shown in Table 3. TABLE 3 0 Inorganic nutrientε added to ultrafiltered whey. MgS0 4 -7E 20 : 3 g MnS0 4 -H 2 0 : 0.1 g K 2 HP0 4 : 4.4 g 5 FeS0 4 /Ξ 2 0 : 0.28 g Whey : 1000 g Concentrated bacterium solution is pumped controllably out of the cultivation system when the bacterium concentration has reached the desired level, for example 60 g dry weight bacterium per liter. The bacterium solution is mixed with Na-alginate solution, for example, Protanal LF 10/60, to an alginate concentration of 0.5-4 (w/v). Preferred con¬ centration iε 1.5$. The bacterium concentration in the alginate beads iε εelected εo as to allow the occurrence of only a minimal diffuεion limitation during the production process. Typical valueε for the bacterium concentration are 1-100 g dry weight bacterium/kg alginate beads. Lactic acid bacteria immobilized in alginate gel were produced as described in Example 1 and Example 2. C 5

Claims: P a t e n t C l a i m ε 1. Continuouε process for production of biocatalyst particles where a biocatalyst (1) iε mixed with hydrocolloid gelling agentε, e.g. , alginate (2) in a mixing chamber (3), the mixed mass is further conducted in a tube (4) to a rotating, perforated cylinder (5), c h a r a c t e r i z e d i n that said cylinder (5) is placed in a reactor (6) and that C the cylinder (5) is rotated at a velocity causing the beads to be hurled directly down into the reactor liquid (11). 2. A method according to claim 1, c h a r a c t e r i z e d i n that the velocity of rotation of the cylinder iε between 2.5 and 6300 m/s 2 , preferably between 2.5 and 360 m/s 2 . 3. A method according to claims 1-2, c h a r a c t e r i z e d i n that the holes in the rotating cylinder vary in number and placement . 4. A method according to claimε 1-3, c h a r a c t e r i z e d i n that the diameter of the holes in the cylinder is from 0.2 mm to 4.0 mm, preferably 0.5-2.5 mm. 5. A method according to claims 1-4, c h a r a c t e r i z e d i n that the biocatalyεt particles produced have a radiuε of from 0.1 to 4.0 mm, preferably 1.0-2.5 mm, and the deviation may be up to 10$. 6. A method according to claimε 1-5, c h a r a c t e r i z e d i n that the proceεε can take place under aεeptic condi¬ tions . 7. A method according to claims 1-6, c h a r a c t e r i z e d i n that there are used biocatalyst particles which do not contain gas bubbles. 8. A method according to claims 1-7, c h a r a c t e r i z e d i n that the biocatalyst particles may contain from 0 to 100 g biocatalyst per kg biocatalyst particle.

Inventors: EIDE OLA, LYSBERG MAGNE, OTERHOLM BJARNE, STORROE IVAR, SVENDSEN HALLVARD, WINNBERG ASGEIR, EIDE, OLA, LYSBERG, MAGNE, OTERHOLM, BJARNE, STORROE, IVAR, SVENDSEN, HALLVARD, WINNBERG, ASGEIR
Applicants: NORSKE MEIERIER
Application year: 1993
Main classification: C12N11
IPC codes: C12M1, C12N11