GB2229649A - Apparatus for the realization of biocatalytic process with solid phase biocatalyst - Google Patents

Description

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APPARATUS IPOR THE REA1IZATION OF BIOCATALYTIC PROCESSES 'iVITH SOLIDPHASE BIOCATAILYST The invention relates to an apparatus for the realization of biocatelytic processes with solidphase biocatalyst.

One of the most dynamically developing areas of the biotechnology is the operations realized with the use of solid-phase biocatalysts. The solidphase biocatalysts are brought about with so-called immobilization technique, in essence to localize the catalytically active biological materials in the reactor space in order to prevent their passing into the flowing phase (J.Chem. Tech.Biotech., 1984, 1Ai 1270. The solid-phase biocatalysts can be produced by immobilizing isolated en- zymes, dead cells, or cell-components. or living cells (microbes$ Plant Or animal cells). In concrete case the most appropriate possibility is selected with regard to the economic and/or stability aspects.

Use of the solid-phase blocatalyst has significant economic benefits compared to the traditional biocatalytiQ procedures, such as:

- continuous or semi-continuous technology is realizable.

- increased volumetric productivity.

- the costs of operation and product recovery are reducible.

In case of production of secondary metabolites with immobilized cell fermentation, further benefit is obtained by reduced viscosity of the ferment liquor due to use of the continuous operating mode, thereby improving the oxygen transfer; certain over-reproduction prob lems can be avoided and loss of the chemically unstable products decreases (Biotechnol.Bioeng. 183. 229 2399-2411) A number of different types of reactor were developed to carry out solid- phase biocatelytic operations On industrial scale the so-called packed- bed reactor is most frequently used. e.g. for glucose izomerization. amino acid resolution or ethanol fermentation (Chem.Eng.

Sci. 1985, 40, 1321-1354.). Fluidized-bed reactors of industrial scale are used for waste water treatment with immobilized biomass (J. 1Valter Pollut. Control Fed. 19779 49 816.). There is an example also for the use 0 of membrane reactor on industrial scale. (Chem.Ing.

Techn. 1988, o, 16-2M.

Several types of the pilot plant and laboratory reactors are known. The most frequently used packed-bdd and fluidized-bed rdactors have been developed In multiunit form (horizontal and vertical cascade construction), among others to prevent enrichment'of C02 produced during the process (J.Chem.Tech.Biotech., 1987, 19, 75-84.), or to reduce axial back-mixing of the substrate solution (Proc. 4th Eur. Congr. Biotech. vol.l. 1987. 273276).

The socalled external and internal loop reactors are used mainly for realization of the immobilized cell fermentation. (Biotech. Bioeng. 1987. 209 498-504; J.Chem.

Tech. Biotechnol. 1986, _6, 415-426.) Although numerous types of reactor exist - as shown in the foregoing - for realization of the solid phase biocatalyzis, fairly many problems connected with reactor technics remain unsolved. primarily in the processes where one of the substurates is gas phase (usually origen). The gasliquid transfer is highly significant in the majority of the traditional biocatelytic systems. e.g. the air-consuming microbes necessitate that the oxygen transfer rate (OTR) should reach the value of 150 mmole/ dm3/h. For efficient.utilization of the useful volume of the apparatus, the mentioned OTR value should be reached with moderately high aeration (1-1.5 vvm). Since the presence of the carrier particles serving for Immobilization of the bioactive materials in most cases deteriorates the gas - liquid mass transfer - the particles grains increase agglomeration of the gas bubbles (Proc. 4th Eur. Congr. Biotech. 19879 vol. 1. 101-104) - in the three-phase fluidization bubble column, or loop systems equipped with traditional gas distributors. max. a fraction of the above mentioned OTR value can be obtained (Chem.Eng.Sci. 1987, 4-2, 543-553; Biotech.Bioeng. 1988, 32, 677-688). Intensification of the gas distribution is rendered more difficult by the relatively low mechanical stability of the major part of the matrix materials, therefore the significant force effects have to be avoided in the catalysis zone. e, 90 a great part of the gels and organic and inorganic materials of high porosity can not endure not only the shear forces associated with the mechanical mixing. but also the force effects induced by the highly turbulent liquid. or liquid-gas jets.

In some fermentation processes connected with microbial plant and animal cell cultures particularly sensitive to infection. further difficulty is tIm t the cells usually entrapped into gel. have to be brought into the fermenter under strile conditions. Namely.the structure of the fermenter generally does not allow - or only with very complicated procedure - in situ the sterile catalyst particle formation.

The invention Is aimed at providing such apparatus for the realization of biocytalytic processes with solid phase biocatelyst, which ensures intensive gasliquid transfer. during the operation of which the catalyst carrier particles of low mechanical stability do not get damaged,, and which Is useful to combine the functions of the gel-type carrier particle forming apparatus and the bioreactor even when special sterilization is required.

The invention Is based on the following recognitions:

If the part of the apparatus providing high specific gas-liquid interfacial area is separated from the part containing the solid phase biocatalyst, the turbulent forces facilitating the gas dispersing do not effect on the catalyst particles, consequently they do not get damaged. Further recognition is that mixing of the gas and liquid phase can be greatly intensified If the mixing takes place in a.circulating pipe. where dispersing elements are built in, and its volume is max. 10 % of the volume of the apparatus catalysis zone. Furthermo-re, by suitably selecting the direction and velocity of the gas-liquid dispersiongs entry into the reaction space brought about in the space of the apparatus used for contact of the gas and liquid phases as well as the structure (formation) of the reaction space. in the latter one the steady circulating flow of the catalyst particles and the extended residence time of the gas bubbles can be ensured. and by appropriate formation of the reaction zone it would be made suitable for coagulation of the sol drops in the reaction zone, - which are produced by the drop-forming unit fitt ing to the apparatus cover. in the period prior to the biocatalysis. said sol drops contain biocata17st (e.g. cell or spore suspension), whereby.the biocatalyst grains In situ are produced in the apparatus under intensified conditions.

On the basis of.these recognitions,, the objective of the invention was accomplished with an apparatus provided with reaction space containing draft tube, a device for charging the catelyzer, supply liquid and gas into the reaction space, as well as a device for discharging the product obtained by the biocatalysis and the gas separated during the process, furthermore a circulating pump. and said apparatus is characterized by having a recirculating pipe the two ends of which leading into the reaction space at points above each other. a filter element is situated before the outlet orifice of the medium treated In the reactor space, where the filter element is used for retaining the solid catalyst carrier particles in the reactor space; the recirculating pump is built into the recirculating pipe dividing it into a pressure and suction branch, and regarding the flow direction of the treated medium, first a flowaccellerator is built into the pressure branch after the pump, then a gas inlet pipe Is leading in, thereafter the pressure branch contains gas dispersing element(s).

According to a preferred embodiment. a phaseseparating chamber is connected on the top of the reactor space provided with a gas outlet stub. and the suction branch of the recirculating pipe starts of the chamber9s lower part and a draft-tube located in the reaction space extends into the chamber.

According to another preferred invention criterion the cylindrical reactor space is closed at the bottom by a similarly cylindrical backing element, in which a duct tangentially joins the circulating pipe's pressure branch. It serves the purpose if the upper surface of the bottom part, the inclined are starting from the bottom of the pressure branch's feed ing orifice extending to the top of this orifice is formed as a frontal surface. and if a drain stub covered with filter element-from the reactor space provided with valve is led through the bottom on the outside. while above the filter element a conical flow-modifying element of upward diminishing cross section is fixed leading Into the interior of the draft-tube.

Such embodiment is also preferred, where the inlet stub of the liquid to be treated leads Into the recirculation pipets pressure branch between the pump and the flow-accelerator element, and the stub discharging 1 the product obtained'--ds'--. ii.^.tesult of the biocatalytic process is leading out from the auction branch of the recirculation pipe.

According to a further invention criterion. the apparatus has a stub leading into the phase-separation chamber for charging the solid biocatalyst material into the reaction space.

A preferred embodiment of the apparatus is characterized by a dropforming head leading into the phase-separation chamber for charging in the biocatalyst material, e.g. bacteria containing sol-drops. which is connected with a tank containing a mixer to receive the sol material. In this case, it serves the purpose If a valve is built in between the dropforming head and the tank, and if the drop-forming head is provided with drop-forming elements tending towards the interior of the phaseseparation chamber.

If density of the solid catalyst particles is lower than that of the liquid phase participating in the biocatalytic process, it is advisable to use that embodiment of the apparatus, the essence of which is that the draft tube built the reaction space conical at the bottom and cylindrical on the top has an upper cylindrical part of greater diameter, and a lower cylindrical part of smaller diameter adjoining the upper cylindrical part with an intermediate conical part.

The apparatus has a conical chamber as continuation downwards of the lower conical part of the reaction space. which is joined by the suction branch of the recirculation pipe. and the pressure branch of the recirculation pipe is tangentially led into the lower range of the draft- tube's upper cylindrical part. and a filter element is arranged before' the orifice of the suction branch leading out from the chamber. In this case, it is beneficial if an auxiliary gas flow inlet nozzle is led through the wall surrounding the tapered chamber at the bottom, which is directed towards the orifice of the draft tube's lower cylindrical part reaching into the chamber.

According to a further invention criterion, the length of the circulating pipe's pressure branch exceeds many times the length of the suction branch. Accordingly. the number of dispergating elements can be increased, and thus intensity of the gas-liquid transfer can be increased.

According to a further embodiment. the apparatus is provided with gas dispersing element(s) possessing confusori diffusor and slot between them. These gas dispersing elements are built first of all into the vertical or near vertical section or sections of the circulating pipe2s pressure branch. The gas dispersing elements with a bar and a surrounding spiral part are arranged in the horizontal or near horizontal section or sections of the pressure branch. The Invention is described in detail with the aid of drawings showing some preferred embodiments of the apparatus given by way of example. and its structural part. in which: Fig. la: Schematic vertical axial section of the apparatus, Fig. lb: Section along A-A marked in Fig. la, Fig. lc: Vertical axial section of a drop-forming element drawn to a larger scale, Fig. ld: Axonometric drawing of a dispersing element drawn to a larger scale, Fig. le: Prontview of another dispergating element, Fig. 2a Schematic vertical axial section of another embodiment of the apparatus, Fig. 2b of the apparatus shown in Fig. 2a, to a Cascade line.

Fig. 3a:Vertical axial section of an element, Fig. 3b.. Section along F-P marked in Fig. 3a.

The apparatus presented in Pig. la - le, is built in the form suitable for entrapping the air-consuming microorganisms. or their spores in gel, and for carrying out quasi-continuous fermentation with microorganismsentrapped in gel (e.g. localization of Brevibacterium flavum on Kcarragenate carrier and 11 its application for the biosynthesis of L-tryptophane).

Main parts of the apparatus are the inlet unit I at the bottom3 the autocirculating reaction unit II above the former one. the phaseseparation unit III, sol drop-forming unit IV adjoining the cover of the former unit, and the forced circulation gas dispersing, unit V, which connects the fluid inlet unit I and phase-Separation unit III by-passing the reaction unit II.

The fluid inlet unit I has a cylindrical bottom element 1, the fluid inlet stub 2 tangentially leading to it from the outside, which - as shown in Fig. lb continues in the tangential duct 3 machined in the bottom element 1. The lower codining surface 5 of space-part 4 (Fig. la), (see also Fig. lb) r;lses in frontal thread form with regard to the longitudinal vertical geometric centre-line x of the apparatus starting from the lower limiting edge of the leading plane 6 and ending at the same plane.

The conical flow-modifying unit 8 is arranged with its peak on the vertical geometric centre-line x spaced above the bottom element 1, and fixed with screw 7 to the bottom element 1. A cylindrical superficies shaped longitudinal filter element 10 is situated between the base-plate of the flow modiftying unit 8 and the upper end plate of the circular (in top view) cent:al part 9 of the bottom element 1 (see also Fig. lb).

Cone angle of the flow-modifying unit 8 is suitably between 50 and 70 0, diameter d k of its base is in proportion to bottom internal diameter Da as (1-1.2) to 2.

Two through going vertical stubs 11. 12 are built into the cylindrical bottom element 1 of the fluid Inlet unit I. Stub 11 is'used for removal of the liquid phase without the catalyst particles (the filter element 10 as seen later - retains these particles), whereas the stub 12 is provided for the comp - lete removal of the liquid-solid suspension from the apparatus. The stubs can be opened with the valves 13 and 14 below the bottom element 1.

The autocirculating reaction unit II has a reaction space 15 surrounded by a cylindrical wall 15a. Internal diameter D, of the house 15a is identical with the diameter D A of the cylindrical bottom element 1. The wall 15a, is surrounded by thermostating mantle 16 spaced from the wall, thus a narrow, circular thermostating space 16 is between the wall and the mantle. A draft tube 17 of diameter d 1 is centrally located in the reaction space 15, in other words, the wall 15a. thermostating mantle 16 and draft tube 17 are concentric with the vertical geometric centre-line z. The diameter D 1 is in proportion to d 1 as 2 to 1. The lower flange of the draft tube extends below the peak -1 V of the conical flow-modifying element 8, i.e. the draft tube partly surrounds the upper part of the flow-modifying element 8. The vertical distance between the lower flange of the draft tube 17 and the d 1 diameter circle cut out on the cone by the vertical projection of the draft tube is determined so, that the imaginary cylinder mantle between this circle and the lower flange of the draft tube and the cross sectional area of the draft tube should be nearly identical.

The phase-separation unit III has a chamber 18 with upwards expanding truncated conical lower part 19 and cylindrical upper part 20 connecting with the conical upper part from above it. The diameter D2 of the upper part is suitably about 1.5-2times the diameter D 1 of the reaction space 15. Weight h2 of the phase-separation unit III is about 1/3-1/4th of the height 1 of the reaction space 15. The conical lower part 19 of the phaseseparation unit III is about 1/3rd of the height of the upper part 20. The total height L of the fluid inlet unit I, reaction unit II and phaseseparating unit III, - i.e. the actual reactor - suitably does not exceed 5-6-times the diameter D, of the reaction space 15.

The wall of the truncated conical lower part 19 is covered inside by a filter element 21 - suitably made of sieve cloth - which has also shape of truncated conical superficies. Mesh size of the filter element 21 is determined so that it should retain the gas to such extent. that It leaves the fluid in the direction of the least resistance into the interior of chamber 18. Liquid. outlet stub 22 and sampling stub 23 lead out from the lower part 19. The vertical draft tube 17 reaches into the Interior of the phase-separation unit's III lower part 19. in particular.with about-0.01 0.02 m above the level determined by the centre-lines of stubs 22 and 23 (the stubs 22 and 23 are suitably horizontal and their centre-lines are in the same horizontal plane).

The upper part 20 of the phase-separation unit III is closed by cover (plate) 24. The cylindrical drop-forming head 25 of the sol drop-forming unit IV is built into the cover 24 symmetrically to the vertical geometric centre-line x. Therefore, the drop7forming head and the lower part 19 and upper part 20 of the phase-separation unit III are concentric with the ver tical geometric centre-line x. The drop-forming head is connected through valve 26 and stub 36 to the tank 27 of the sol drop- forming unit IV. Gas outlet stub 28 leads out from cover 24, which is connected with cooler 29. Diameter D.. of the drop-forming head 25 is about half of the diameter D1 of the reaction space 15. The 1:

- drop-forming head 25 may also be surrounded by thermostating mantle (not shown), and its volume is about l/loth of the tankgz 27 (sol tank) solume. which is part of the sol drop-f ordiing unit IV.

The tank 27 conical at the bottom and cylindrical on the top - concentric with the vertical geo metric centre-line x - is closed by cover 27a, and inoculating stub 30, filling stub 31 (filling orifice), and stub 34 lead through this cover from the tank. and a tube for pressurized air flow is connected with stub 34 (not shown). The wall of tank 27 is marked with reference number 27b, which is surrounded by thermostating mantle 27c spaced from the former one, which confines with wall 27b a narrow thermostating space 27d. The volume of tank 27 is about 50 % of the reaction spaces 15 volume of the reaction unit II, A mixing propeller 33 reaches into the tank 27. in particular, into its truncated conical lower part, the connecting shaft of which is in the vertical geometric centre-line x and driven by motor M, Drop-forming elements 35 are built into the base of the drop-forming head 25. one of them is shown in section and drawn to an increased scale in Pig. lc. Filter 36 - made of sieve cloth suitably with 0.1 mm mesh size - is built Into the drop-forming heed 25 above the base. The number of drop- forming elements 35 concentrically distributed on the base. can be deter- mined with the following empirical formula:

00 Dl(m) 2 19 - n = - 2 6 The hole diameter d f-of the drop-forming elements 35 (Fig. lc) is 0.3-0. 5 mm depending on the character of the sol.

The forced circulating gas dispersing unit V of the apparatus has a circulation pipe 50, one end of which is connected with stub 2 of the fluid inlet unit I, the other end with liquid outlet stub 22 of the phase-separation unit III. The circulation pipe 50 (max. diameter of it is about 1/4 - 116th of diameter D, of the reactor space 15) has two sections:

its suction branch extends from the liquid outlet stub 22 to the circulating Pump 38 of variable delivery output, and its pressure branch 39 leads from the Pump 38 to the fluid inlet stub 2. The location of Pump 38 is selected in the circulation Pipe 50 so that it is possibly closest to the stub 22. i.e. the pressure branch 39 of the circulation pipe 50 should be as long as possible. The suction branch and the pressure branch 39 can be short circuited with valve 41 built into the by-pass pipe 40.

The flow-accelerating element 42 built into the pressure branch -359. suitably confusor serves for high speed acceleration of the flow rate. Nozzle 43 is provided for the steady gas flow located in the 1.

pipe 44 leading into the pressure branch 39 after confusor 42 regarding the direction of the medium flow marked with arrow g, said pipe 44 serves for inlet of the gas, generally air (oxygen), in the direction of arrow p (Fig. la) required for the biochemical reaction. Dispersing elements 45 are built into the pressure branch 39 between pipe 44 and stub 2, one of them is presented in the axonometric drawing of Fig. 1d drawn to an increased scale, and these elements provide the intensive gas-liquid mixing and d-ispersation of the gas phase to fine bubbles. The dispersating element 45 shown in Fig. le and ld has a contracted slot in the centre, the fore-end of which Is connected with the confusor. and the other end with a diffusor. regarding the direction of the medium flow marked with arrow g (Fig. la), (or slot 45a - as shown in Fig. ld is connected partly with the confusor 45b, and partly with the diffusor 45c). The slot type elements are built into the vertical pipe tract if the gasliquid flows downwards. The dispersating element 46 shown in Fig. le is formed by a straight bar 46a and its ourrounding spiral part 46b; the dispersing element 46 should be built into a horizontal pipe tract. The dis persing elements 45 and 46 are replaceable and their characteristic dimensions can be determined according to the air consumption of the given cell-culture and dimensions of the reactor.

The stub 48 serving for inlet of the supply solution (arrow p. Fig. la) and stub 49 for removal of the product (arrow t, Fig. le) are parts of gas dispersing unit V with forced circulation which can be closed ar opened with valves 47a and 47b.

The above described apparatus functions as f ollows:

The tank 27 of the sol drop-forming unit IV is filled up through the filling inlet with Na-alginate sol of appropriate concentration while the valve 26 is in closed position, then the filling inlet is closed and the whole reactor (bioreactor) is sterilized with the generally used steam sterilization method. To carry out this operation. the steam Is admitted through stub 44 of the dispersing unit V, which is taken off the system through stub 38 leading out from the chamber 18 of the phase-separation unit III. The tank 27 of the sol drop-forming unit 1V and the dropforming head 25 are sterilized with 120 0 0 steam admitted into the thermostating space 27d, meanwhile the solution in the tank 27 is mixed with mixing propeller 33 to improve the heat transfer. Upon completion of the sterilization, the reactor space and circulation pipe 50 are dewatered through stub 12 connecting with the fluid inlet unit I. then the reactor and sol solution are cooled to room temperature with cooling water circulated in the thermostating space 16a and thermostating space 27d respectively.

In the next step the reactor space 15 and partly the chamber 18 - are filled up to the height of the draft tubegs 17 upper flange with sterile CaC12 precipitating solution using the pump 38. which is fed into the system through stub 48, The sol solution in the tank is inoculated through the inoculating stub 30 with Brevibacterium flavum cell-suspension. and the inoculated solution is homogenized with mixing propeller 33 until the homogeneous cell - concentration is obtained. Then the homogenized sol-cell suspension obtained by opening the valve 26 is fed from the tank 27 into the cylindrical drop-forming head 24 of the sol drop-forming unit III. Here the filter 36 retains the coarse micelles incidentally present in the suspension, while the suspension itself flows into.the drop-forming elements 35. The suspension flows down through stub 34 as a result of the sterile pressurized air flow fed into the tank 27s the flow velocity is controlled by varying the air pressure The drop-forming unit 25 is suitable for feeding cell/sol suspension at max.

w 1 0.4 n (dm3/h) flow rate (n = number of drop-forming elements 35).

According to the formula for instance with a 0.2 m diameter reactor -20 dm31h feed rate can be reached, and this value approaches the output of the known vibration sol drop-forming units with the highest output used in pilot plant size. but contrary to those. it does not disintegrate the cells.

Drops of the cell/sol suspension fall from the drop-forming head 25 into the 0a012 precipitating solution being in the reactor space 159 where they soliC,fy to gel, and settle through the draft.tube 17 to the bottom of the reactor. During the time of the gel particle formation sterile air flow is through pipe 44 and built-in nozzle 43 at a volumetric velocity corresponding to 1 cm/s linear velocity relating to the empty apparatus cross section. through circulat ion pipe 50, stub 2 and duct 3 (Fig. lb) into the reactor. Under the influence of the tangentially fed liquid-air jet inducing spiral upward flow starting from the frontal surface 5 (Fig. lb). the gel particles settled on the bottom element 1 together with the CaC12 solution rise in the space of cylindrical a=ulus between the wall of the reaction space 15a and the draft tube 17, then reaching the upper flange of the dxft tube 17 and-falling over it inwards, they sink down again. and this way the gel particles circulate in the reactor space 15. The flow conditions induced by sterile 1 21 - air flow improves the solid-liquid Mass transfer. i.e. diffusion of the Ca2+ions into the gel grainag thus they are gelled along their whole cross section in a shorter time - compared to the known methods consequently the cells closed in the gel are less exposed to the possibly damaging effect of the Ca012 solution.

After completion of the gel particle formation. the valve 26 is closed, and overpressure exerted to the interior of the tank 27 is stopped through stub 34, the valve 13 is opened and through stub 11 the CaC12 solution is taken off from the apparatus, then while the autvocirculation valve 41 Is in open position. The sterile culture media into the reactor is fed by Pump 38. (If necessary. before admitting the culture media, the gel particles are washed over with sterile distilled water). The culture media is fed through stub 48 into the circulation pipe 50. As soon as the level of the culture media reaches the upper flange of the draft tube -17, charging of the culture media is stopped, the valve 41 is closed. but the culture media is further circulated with PUMP 38. Through pipe 44 and nozzle 43 (the latter one serves to stabilize the gas flow rate) sterile air flow is charged into the pressure branch 39 of the circulation pipe 50 (arrow p, Fig. la) at a flow rate that provides the air consumption of the bacterium culture at the ventillatIon rate of 1 - 1.5 vvm. In case of air-consuming microorganisms, it is desirable to keep the value of the oxygen transfer rate (OTR) in the range of 150-250 mmole 02/clm 3 h. This OTR interval in the apparatus can be reached by setting the pump 38 circulated liquid flow rate to 50-80 % of the actual volumetric flow rate of the gas (sterile air flow). Under the influence of the highly turbulent gas-liquid flow very fine gas- liquid dispersion is produced in the dispersing elements 45 and 46 built into the circulation pipe 50 (see Fig. ld and le). and renewal of the interfacial area will be extremely intensive. The pressure drop in the dispersing elements 45, 46 does not exceed (0.8 - 1.0) 105pa.

The gas-liquid dispersion enters the reactor's bottom element 1 through the fluid inlet stub 2 and tangential duct 3 (Fig. lb)g and - due to the frontal thread shaped surface 5 - it is.forced to upward spiral motion.The medium containing liquid and gas phase entering with 10-20 m/s velocity induces fluid motion rising on spiral path in the reactor space 15 in the cylindrical annulus. located on the outside of the draft tube 17. The gas bubbles - agglomerating while rising- move on a spiral path, thereby the average residence time of the. bubbles in the reactor space will be longer. on the other hand, as a result of the forces 1 r, ,S 4_ of different directions, such shear stresses will occur. which disrupt the already agglomerated bubbles. thus increase the specific gas-liquid interfacial area in the reactor space 15.

The gas-liquid flow carry along the catalyst carrier gel particles, and they rise. to the phaseseparation unit III above the flange of the draft tube 17, then through the draft tube together with the autocirculating flow induced by gas bubbles flow back to the bottom of the reactor space 15. Flow of the gel particles (solid phase biocatalyst) and the liquid phase is regulated by the flow-modifying element 8. and in addition, it prevents the formation of dead space in the apparatus. The fluid flowing down through the draft tube 17 entrains the finaly distributed gas bubbles as well.

The gas emerges from the liquid phase in the phase -se pa ra I'Jion unit III, and it leaves the apparatus through stub 28. The liquid flow leaves the phase-separation unit III through stub 22 but it can flow into stub 22 only through filter 21, which retains the biocatalyst particles (gel particles). The liquid phase flows from the stub 22 Into the suction branch 37 of the circulating,pipe 50,, and from there through the PUMP 38 into the pressure branch 39.

The fermentation process and condition of the culture media can be followed with attention by x - Z4 - analysing the samples taken through stub 23 leading out from the phase- separation unit III. In the productive stage of the fermentation the continuous take-o.LI'f of the product and continuous feed of the culture media can be accomplished with the pump or pumps connected to stubs 48 andjor 49. or with overflow.

If replacement of the culture media is necessary, the ferment liquid is drained through stub 11 by 0Dening the valve 13 (Fig. la). and the reactor is filled up with fresh culture media as described in detail earlier.

After completion of the -&'#-otal fermentation cycle the ferment liquor together with the catalyst carrier gel particles is drained through stub 12 by opening the valve 14. then sterilized with steam.

The embodiment of the apparatus according to Fig. 2a, 2b - apart from lack of the sol drop-forming unit - is virtually the same as in Fig. la- le. hence the earlier reference numbers are used for marking the identical parts of the apparatus. The apparatus according to Fig. 2a, 2b is built in the form suitable for the realization of quasi-con,#liinuous fermentation with microorganisms immobilized on the pre-formed carrier material. the density of which is less then the density of the fluid. A specific feature of the process is that it is anaerobic, or requires only a little airg at the same time the gas (e.g. carbondioxide) as by-product inhibits the fermentation e.g. the ethanol fermentation with Saccharomyces,cells -, therefore, inert gas (e.g. nitrogen). or mixture of inert gas and air is used to displace 002 from the liquid phase. Further characteristic of the process is, tIm t the product also has fermentation-inhibiting effect, therefore. in order to reduce the backmizing of the fluid. several apparatuses are operated in cascade arrangement.

As mentioned before,, the fluid inlet unit I of the apparatus according to Fig. 2a differs from the unit according to Fig. la in that it is provided ivith thermostat-ing mantle 51 surrounding the narrow thermostating space 51a. Stub 52 for charging through the cover into the chamber 18 is connected to the phase-separation unit-III instead of the sol drop-forming unit. where the charging takes place with level control. The cascade elements can be connected with such pipe and valve system, whereby their sequence is variable during operation. Members (reactors) K, - K 3 of the cascade reactor (cascade line) are connected with pipes 60, 61.

The apparatus according to Fig. 2a, 2b functions as follows:

Members K, - K 3 of the cascade line (11411g. 2b) 1 - 2 6 - are filled up with culture media using the pumps 57-59. Then the valves 47b are closed and the reactors of the cascade line are sterilized according to the generally known sterilization method of the fermenters. After completion of the sterilization. the reactors are cooled down, then the.fermentation temperature is set. and the biocatalyst particles containing immobilized Saccharomyces cells are fed thwaugh the charging stub 52 into the sterilized, culture media, and the level of the culture media is set to the level of the draft tube9s 17 upper flange (Pig. 2a).

In the next step. sterile inert gas or the mixture of inert gas and air is fed through stub 44 containing the nozzle 43 into the members K 1 - K 3 of the cascade reactor at a flow rate sufficient to displace the 002 produced during fermentation, and to ensure autocirculation of the liquid/solid suspension in the reactor space. In order-to ensure the Intensive gas-liquid mass transfer, the liquid is circulated with circulating pump 38 by each cascade member through dispersing elements 45 and 46 in the circulation pipe 50 (see also those said in connection w- lth Pig. la-le.) The gas containing 002 in each member (reactor) K1-K3 emerges from the liquid phase in chamber 18 of the phase-separation unit III, and it leaves the reactor through stub 55. and the liquid leaves the reactor space 15 through filter 21 and stub 22 (the filter 21 retains l - the catalyzer grains). and circulates through the suction branch 37 of the circulation pipe 50 into the pump 38, and from there into the pressure branch 39.

After reaching a predetermined number of cells. continuous charging of the sterile culture media is started with pumps 57-59 (Fig. 2b) and the product is continuously taken off.

Embodiment of the apparatus according to theinvent-ion shown in Fig. 3a and 3b is used for the reali zation of the biochemical reaction catalyzed with fixed enzyme, one substrate of which is in liquid phase. the othe-substrate is in gas phase, and the enzyme is immobilized on small carrier particles of low density sensitive to deformation. Such reaction is for example the oxidation of glucose to gluconic acid in the presence of glucose-oxidase immobilized on polyacrylemide bead polymer.

Dlain parts of the apparatus are the fluid outlet unit V!, reaction unit II, and the gas dispersing unit V with forced circulation.

The fluid outlet unit V1 has an upward expanding truncated conical chamber 64, from which fluid outlet stub 64 is leading out, and a stub 65 for feed of the auxiliary gas is leading in. 1Por removal of the liquid from the apparatus without catalyst carrier particles a stub 67 with valve 66. and for removal of the total reactor charge a stub 69 with valve 68 are provided. The orifices of stubs 64. 67 and 69 leading into the chamber 63 are separated from the interior of the chamber by a truncated conical filter element made of sieve cloth located on the inner surface of 'Ghe chamber. Mesh size of the filter element 70 is determined as to exert such resistance for the gas phase, that it rises in the reactor space in the direction of least resistance. The stub 65 for inlet of the auxiliary gas flow is connected with nozzle 71 reaching to the interior of the chamber 63 by which a stead7 gas flow is attained. Cone angle of the chamber is suitably between 50 and 700. its mean diameter B. may be about 1/3rd of the reactor's diameter B,. and its height may be about 1/5th of the reactor's full height L.

Autocirculation reaction unit II is connected to the fluid outlet unit V! provided with upward expanding truncated conical lower part 73 and cylindrical upper Part 74. The lower part 73 can be regarded as continuation of chamber 63. Height of the lower part 73 is about in proportion to the height of the UPPer Part as 1 to 3. Full height L of the reactor (built together with the fluid outlet unit V1 and reaction unit II) is not greater than 1.8-2.5- times the diameter 31 of the reaction space 75 (cylindrical upper Part 74). In the vertical geometric centreline x, of the reactor is the draft tube 76 consisting of three parts: the lower cylindrical part 77 of smaller diameter el.the upper cylindrical part 78 of greater diameter e 3 and between them the upward expanding truncated conical intermediate Part 799 the length of which are marked starting from the bottom with reference letters hl, h2, h3 respectively. It is advisable if cl: C2: C3 t 3 1: 3 and 1: 4 9 f urthermare B, 0; 1: 2.

el: e2 = e3 Height H, of the reaction space 75 is the same or nearly the same as the h_eight of the draft tube 76.

but the lower end of the draft tubegs 77 lower cylind rical part reaches into the inluerior of the chamber 63, and it is located with about spacing Ba/3 above the peak of nozzle 71 reaching into the chamber. The fluid outlet unit VI and reaction unit II are surrounded by thermostating mantle 80a enclosing the thermostating space 80.

Fluid charging stub 81 is tangentially lead ing to the upper cylindrical part of the draft tube 76 in about the lower quarter of height c3, conducted through the tank's 72 wall (and thermostating mantle 80a), The stub 81 is suitably horizontal and its diameter is about 1/10th of the diameter e 3 of the upper cylindrical Part 78.

The reaction space 75 is closed with cover 82, through which the gas outlet stub 83 is leading out from the reaction space. and the catalyst charging stub 84 is leading into it.

One end of the circulating pipe 85 is connected to stub 64 of.the fluid outlet unit VI, and its other end to the fluid charging stub 81 of the reactor space, with dispersing elements 45. 46 built into the circulation pipe 85 according to Pig. ld and le. The circulation pipe 85 has a suction branch 87 extending from stub 64 o the circulating pump 861 and a pressure branch 88 extending from the pump 86 to the fluid charging stub 81. A confusor 89 is built into the latter one. to considerably increase the velocity of the flow rate. Stub 92 with nozzle 91 is provided for feeding in the gas flow. The dispersing elements 45 are in the vertical tract of the pressure branch 88 between the confusor 89 and stub 81 (Fig. ld), and the dispersing elements 46 are built into the horizontal tract (16lig. le); such dispersing element 46 is located in stub 81 as clearly shown in Fig. 3b.

A stub 93 with valve 94 is leading into the suction branch 87 before stub 64. and the flow-accelerating element 89 preferably Pipe 95 with valve 96 before the confusor serving for feed of the substrate.

The apparatus according to Fig. 3a and 3b functions as follows:

The chemically sterilized apparatus is filled up through the catalyst charging stub 84 with polyacrylamide bead-immobilized enzyme suspended in glucose substxate. The volume of the immobilized enzyme charge is about 30-40% of the reactor's useful volume. Then glucose substrate solution is fed through stub 95 according to arrow'o into the pressure branch 88 of the circulating pipe 85 with the aid of pump 86 and by opening the valve 96. The glucose substrate solution is fed through pipe 81 into the reaction space 75. The feed is continued until the liquid level reaches the upper flange of casing pipe 76. At this pointg the valve 94 is closed.

After setting the reaction temperature with the liquid circulated in the thermostating space 80, the auxiliary air flow inducing autocirculation of the liquid phase and catalyst particles is started through the auxiliary inlet stub 65 and nozzle 71. The volumetric velocity of the auxiliary air flow is equal to about 10% of the volumetric velocity of the air flow (charged through stub 92) providing the oxygen consumption of the reaction. The bubbles rising from the nozzle 71 Passing into the lower cylindrical Part 77 of the draft tube 76 exert suction effect to the liquid phase and to the biocatalyst particles of lower density then that of the liquid suspended in the liquid phase. This way, upward fluid and particle movement 1 takes place in the draft tube 76, and it recirculates through the annulus between the outer well of the draft tube 76 and inner face of the tank 72 wall. Intensive agglomeration of the bubbles takes place in the draft tube 76, thus the auxiliary air flow is virtually insignificant as oxygen source, it serves only for regulation of the particle circulation.

The required oxygen concentration is provided in the liquid phase in such a way, that forced circulation is realized in the circulating pipe 85 with the aid of pump 86: the pump 86 dravis the liquid through the fluid outlet stub 64. while the filter element 70 retains the catalyst particles in the chamber 63. The pump 86 feeds liquid into the pressure branch 88, mixing it there wi-,jh the air flow fed in according to arrow p through stub 92 and nozzle 91. and where the dispersing elements 45, 46 as described in connection with.2ig. la - le - provide the required oxygen transport from the gas phase to the liquid phase.

The gas-liquid dispersion flows from the pressure branch 88 through the fluid charging pipe 81 into the upper cylindrical part 78 of the reactor's draft tube 76. then the liquid steps over its flange and together with the catalyst particles moving down between the tank 72 wall and outer surface of the draft tube 76 returns to the bottom of the reactor. the gas leaves the reactor through stub 83.

Significance of the tangential fluid inlet through pipe 81 is found in the fact, that the substrate solution with high oxygen content moving on a spiral path is in intensive contact with the catalyst particles in the draft tube for elongated time, thus ensuring the proper conversion.

The continuous operation mode can be realized with feed of the substrate solution in the direction of arrow o under the open position of valve 96 and by product take-off in the direction of arrow t through stub 93 while the valve 94 is In open position. or with cascade connection of several reactors.

In case of bach operation mode of the reactor, the product is discharged from chamber 63 through stub 67 by opening the valve 66. The reactor is comp- let.ely drained through stub 69 by opening the valve 68.

Advantages of the invention can be summed up as follows:

A fundamentally important advantage of the apparatus is that carrying out the high specific oxygenconsuming biocatalytic operations in the presence of solid phase biocatalyst Is made economically possible by maximal utilization of the benefits given by application of the solid phase blocatalyst, at the same time. the mass transfer difficulties arising in the traditional methods are eliminated by intensifying the gas-liquid 1 contact. and by the stable circulation of the biocatalyst immobilized on the carrier particles. Further significant advantage is that - because the reaction space in the apparatus is separated from the space of 'the intensive gas-liquid contact - the catalyst particles are not exposed to high mechanical stresses. thus do not get damaged during the biocatelytic operation.

If cell cultures especially sensitive to infection are used as biocatalyst. a preferred version of the apparatus offers the possibility to entrap the cells in situ in gel. i.e. to produce the solid biocatalyst beads in the apparatus itself under more intensive and safer conditions then offered by the known methods.

Further advantage is that by feed and intensive distribution of inert gas (or the mixture of inert gas and air), the opportunity is open to displace the damaging gaseous by-products from-the liquid phase of the fermenter under economical use of the inert gas.

Finally an important advantage is tba t if the apparatus is used as a member of the cascade 2te, such biocatalytic processes can also be carried out with high efficiency, where the product and/or gas as by- product (e.g..carbon-d:Loxide) inhibit the biocatelytic process (e.g. fermentation).

Naturally the invention is not restricted to the embodiments of the apparatus described In detail 1 in the foregoingg but it can be realized in many ways within the protective circle defined by the claim points.

Claims (15)

What we claim is

1. Apparatus for the realization of biocatalytic process with solid phase biocatalyst. said apparatus is provided with reaction space containing draft tJube,, a device for charging the catalyst. cul ture media and gas into the reaction space. as well as with a device for discharging the product obtained by the biocatalysis and the gas separated during the process, furthermore with a circulating pump. characterized by possessing a recirculating pipe (50,85) its two ends leading into the reaction space (15,75) at points above one another; in which a filter element (21,70) is arranged for retaining the solid catalystcarriers in the reactor space (15,75) before the outlet orilfice serving for discharge of the treated medium from the reactor space (15,75); a recirculating pump (38v86) is built into the recirculating pipe (85) divided into pressure branch (39, 88) and suction branch (37987) and a flow-accelerating element (42.89) is built into the pressure branch (39.88) after the pump. regarding the flow direction (g) of the treated medium, then a gas inlet pipe (44,92) is leading in; furthermore the pressure branch (39,88) contains gas. dispersing elements (45.46), Ilt".qlig. lep 2a and 3a).

2. Apparatus according to claim L. characterized by a phase-separation chamber (18) connected to the reactor space (15) provided with gas outlet stub (28,55), and suction branch (37) of the circulating pipe (50) starting out from the lower part of this.chamber (18).intowhich a draft tube (17) reaches located in the reaction space (15), (Fig. leg 2a).

3. Apparatus according to claim 1 or 2. characterized by a cylindrical reactor space (15) closed at the bottom with a similarly cylindrical bottom element (1) which has a duct (3) tangentially adjoining the inflow orifice of the circulating pipe's (50) pressure branch (39), (Fig. la and lb).

4. Apparatus according to claim 3. characterized by an upper face (5) of the bottom element (1) formed as an inclined curved - frontal thread surface starting from the bottom of the pressure branch (39) inflow orifice and extending to the top of this orif ice ( Fig. la and lb).

5. Apparatus according to claim 3 or 49 characterized by a drain stub (11) conducted through the bottom element (1) provided with valve (13) on the outside and covered with filter element (10) partitioni.ng from the reactor space on the top, and a conical flow-modifying element (8) of upward reducing cross section reaching into the interior of the draft tube (17) is fixed above the filter element (10), (Fig. la and 2a)

6. Apparatus according to any Of Claims 1-5, characterized by stub (48. 95) for feed in of the liquid to be treated that leads into the pressure branch (39.88) of the circulation pipe (50,.85).between the pump (38.86) and flow-accelerating element (42989), and stub (49.93) for discharge of the product obtained as a result of the biocatalytic process leads out from the suction branch (37.87) of the circulation pipe (50), (Fig. la, 2a, 3a).

7. Apparatus according to any of claims 1-6. characterized by a stub (52) leading into the phase-separation chamber (18) for feed of the solid biocatalyst material into the reaction space (15), (Fig. 2a).

8. Apparatus according to any of claims 16, characterized by a dropforming heed (25) leading into the phase-separation chamber (18) for charging in the biocatalytic material, e.g. sol drops entrapping in bacteria,, which is in connection with a tank (27) to receive the sol material and said tank contains a mixer (Fig. la),

9. Apparatus according to claim 8. characterized by a valve (26) built in between the dropforming head (25) and tank (27), (Fig. la).

10. Apparatus according to claim 8 or 99 characterized by the dropforming head (25) possessing 39 drop-forming elements reaching toward the interior of the phase- separation chamber (18), (Fig. la and lc).

11. Apparatus according to any o4E claims 1-6. characterized by the draft tube (17).reaching below a conical. above cylindrical reaction space (75) possessing a greater diameter (e 3) upper cylindrical part (78) and a smaller diam er (e 1 cylindrical part adjoining the upper part with an intermediate conical part (79); the apparatus has a conical chamber (63) as continuation downwards of the conical lower part (73) of the reaction space (75) connected with the auction branch (87) of the circulating pipe (85), and the pressure branch (88) of the circulating pipe (85) is tangentially conducted in the lower range of the draft tubega (76) upper cylindrical part (78)q and a filter element (70) Is arranged before the orifice of the suction branch (87) leading out from the chamber (63), (Fig. 3ag 3b).

12. Apparatus according to claim 11. characterized by an auxiliary gas flow nozzle (71) conducted through the well surrounding the conical chamber (63) at the bottom is directed to the orifice of the lower cylindrical part (77) of the draft tube (76) reaching into the chamber (63), C1Pig. 3a).

13. Apparatus according to any of claims 1-12, characterized by the length of the pressure branch - 40 (39,88) of the circulating pipe (50.85) exceeding many times the length of the suction branch (37,88).

14. Apparatus according to any of claims 1-1-3, characterized by having gas dispersing elements (45) possessing a confusor (45b). diffusor (45c) and interconnecting slot (45a). (Fig. ld).

15. Apparatus according to any of claims 1-14. characterized by having gas dispersing element(s) (46) vrith a straight bar (46a) and surrounding spiral part (46b) (Fig. le).

Published 1990 a.. The Patent Office. State:ouse- 66 71 iligi, Holborn. London WC IR 4TP- Purther copies maybe obtained from The Patent Office Sales Sranch, St Mary Cray. Orpington. Kent BR5 3RD. Printed by Multiplex techniques ltd. St Mary Cray, Kent. Con. V87