AU620802B2

AU620802B2 - Process for the continuous fermentation of media containing carbohydrate, aided by bacteria

Info

Publication number: AU620802B2
Application number: AU13934/88A
Authority: AU
Inventors: Wolfgang Salzbrunn; Alfred Schmidt; Eva Steiner
Original assignee: Vogelbusch GmbH
Current assignee: Vogelbusch GmbH
Priority date: 1987-03-10
Filing date: 1988-03-10
Publication date: 1992-02-27
Anticipated expiration: 2008-03-10
Also published as: CN88101266A; EP0305434B1; EG18209A; ATA56687A; DE3853934D1; EP0282474A1; HUT52161A; KR890700670A; ES2076163T3; EP0305434A1; DD280789A5; JPH01502479A; HU203786B; ZA881671B; NZ223794A; WO1988007090A1; FI894251A0; ATE123530T1; AT388174B; AU1393488A

Abstract

Process for the fermentation of media containing carbohydrate, aided by bacteria forming butanol, acetone, ethanol and/or isopropanol, carried out in at least two stages, the first consisting essentially of continuous cultivation of the bacteria, the second, carried out continuously or in batches, consisting essentially of product formation. In order to achieve long-duration stability of the process, as well as an increase in productivity and in sugar break-down, the bacteria are immobilized during the second stage on a carrier material. Medium can therefore be drawn off at the product formation stage and the formed product can be isolated, thus avoiding inhibition caused by too high a concentration of the product. The device for implementing said process consists of a bioreactor, preferably an agitation pan bioreactor (4), and a reactor provided with a bacteria-retentive material, preferably a fixed-bed reactor (7), disposed successively. An external loop may be attached to the fixed-bed reactor (7) for product isolation, preferably continuous.

Description

See reverse side of this form for guidance in ,compieting this part Applicant is the assignee of the saidL 1nvenTuL::'.

4. he asi Aplication(Qo referred to in paragraph 2 of this Declaration was/warj4~ hc first Aplction~t) made in a Convention country in respect of the invention, the subject of the Application.

DECLARED at November, 188 BERICHTIGTE -in WO 88/07090 IC12P 7/061 (No. 2111988). under INID Numbers (71X(72) "Applicat et Inventor". replac', FASSUNG* "!(ror all designated States except AT AU BE CH DE FR GB IT JP LU NL SE US)" by -(fr all designatOi States except AT AU BE CH DE FRGB ITJP LU NL S) S INTERNATIONALE ANMEfi'N E FN H DEM VERTRAG tIBER DIE 4. ITERNTIONLE USA FAI~.~IBLDES PATENTWESENS (PCT) (51) Internationale Patentklassifikation 4 (11) Internationale Verbffentlichungsnummer: WO088/ 07090 C12P 7/06, 7/16, 7/26 Al (43) Internationales Verbffcntlichungsdafum: 22. September 1988 (22.09.88) (21) linternatienalles Aktenzeichen: PCT/AT88/000 12 A-2700 Wr. Neustadt (AT).

(22j Internatienalles Anrieldedatum: 10. Mdrz 1988 (10.03.88) (74) Anwalte: ITZE, Peter; Amrerlingstralle 8, (AT) usw.

(31) Prieritatsaktenzeichen: A 566/87 (81) Bestinmungsstaaten:.AT (europaiiisches Patent), AUJ,' BB, BE (europ~isches Patent), BG, BR, CH (europai- (32) Prierititsdatum: 10. M~rz 1987 (10.03.87) sches Patent), DE (europdisches Patent), DK, FI, FR (33) Prioritatsland: AT (europdisches Patent), Gb (europ~isches Patent), H-U, IT (juropgisches Patent), IP, KP, KR, LK, LU (euro.

(71) Anmelder (nur far A TA U BE CH DE FR GB IT JP LU p~iiches Patent), MC, MG, MW, NL (europdisches Pa- NL SE): VOGELBUSCH GESELLSCHAFT M. B.H. tent), NO, RO, SD, SE (europdisches Patent), SU, US& [AT/AT]; Blechturingasse 11, A- 1050 Wien eifntch (71)(72) Anmelder und Erfinder (far alle Bestimmun essmaten Mit internationa lern Recherchen bericht.

(ausser ATA UBE CH DE FR GB IT JO L UNL VoT- A blauf der far 4nderungen der A nspriiche zugelas- EFFENBERGER, Helmut [AT/All; FlorianiZg- senen Frisi. Veriffenzdichung wird wiederholt fallsdAn-.

asse 38/13, A-1080 Wien derungen eintreffen.

(72) Erfinder;und Erfinder/Anmelder (nur far US) :SCHMIDT, Alfred [AT/AT]; Pacassistrage 29, A-I 130 Wien STEI- NER, Eva [AT/All; Kleinengersdorferstr. 8, A-2100 Korneuburg SALZBRUNN, Wolfgang [AT/ AT]; Briiunlichgasse 11, (54) Title: PROCESS FOR THE CONTINUOUS FERMENTATION OF MEDIA CONTAINING CARBOHYDRA- TE, AIDED BY BACTERIA (54) Bezeichnung: VERFAHREN ZUR KONTINU IERLICH EN VERGARUNG VON KOHLENHYDRATHALT[- GEN MEEDIEN MIT HILFE VON BAKTERIEN (S7) Abstract RErN Process for the fermentation of media containing carbohydrate, aided- by bacteria forming butanol, acetone, ethanol and/or isopropanol, carried hI out in at least two stages, the first consisting essentially of contiruous cuti s vation of the bacteria, the second, carried owt continuously or in batches, consisting essentially of product formation. In order to achieve long-dura-.IPOUT tion stability of the process, as well as an increase in productivity and in sugar break-down, the bacteria are immobilied during the second stage on a carrier material. Medium can therefore be drawn off at the proCuct formation stage and th~e formed product can be isolated, thus avoiding inhibition caused by too high a concentration of the product. The device for implementing said process consists of a bioreactor, preferably an agitation pan bioreactor and a reactor provided with a bacteria-retentive material, preferably a fixed-bed reactor disposed successively. An external loop may be attached to the fixed-bed reactor for product isolation, preferably continuous.

(57) Zusamnienfassung Das Verfahren zur Verglirung von kohlenhydrathliltigen Medien mit Hilfe von Butanol, Aceton, Ethanol und/oder Isopropanol bildenden Bakterien, wird wenigstens zweistufig ausgefiffhrt, wobei im ersten Verfahrensabschnitt im wesentlichen die Bakterien kontinuierlich kultiviert und im zweiten kontinuierlicn oder chargenweise gefiffhrten Verfahrensabschnitt im wesentlichen das Produkt gebildet wird. Zwecks Erzielung einer Lanigzeitstabilit~ft des Verfahrens und einer erh6hten Produktivit~t sowie eines erhbhten Zuckerabbaues werden die Bakterien im zweiten Verfahrensabschnitt an einem Triigermaterial immobilisiert. Dabei, kann aus der Produktbildungsstufe Medium abgezogen und das gebildete Produkt abgetrennt werden, um eine Inhibierung durch zu hohe Produktkonzentration zu vermeiden. In einer Voricng zur Durchfuhrung dieses Verfabrens sind emn Bioreaktor, vorzugsweise emn Riihrkesselbioreaktor und emn rit Mikroorganismen riickhaltendem Material besttlckter Reaktor, vorzu3sweise eim Festbettreaktor hintereinanderges(,haltet, wobei gegebenenfalls an den Festbettreaktor eine externe Schlaufe zur, vorzugsweise kontinuierlichen, Produktabtrennung angeschlossen ist.

*(Siehe PCT Gazette rNr. 24/1988, "Section 11") Ii la A PROCESS FOR THE CONTINUOUS FERMENTATION OF CARBO- HYDRATE-CONTAINING MEDIA BY MEANS OF BACTERIA.

The subject of the present invention is a process for the fermentation of carbohydrate-containing media by means of bacteria forming butanol, acetone, ethanol and/or isopropanol, the process being carried out in at least two stages: basically, in the first stage the bacteria are being cultivated substantially continuously, and in the second stage, which is conducted continuously, Sor in batches, primarily the product is being formed.

A process of this kind exists in which two 10 conventional growing stages are provided and the course 0 0 of the process is governed by controlling the rate of flow and by the aid of phosphate limitation, so that in the first stage (growth stage) the bacteria-concentration remains high, i.e. steady-state flow is attained and that a portion of the carrier is already converted into butanol, acetone and/or ethanol. In the second stage, o"S ao then, a yield as high as possible in the fermentation 6o products sought is to be obtained, in as short a time as possible. Should an excessive rate of flow be chosen in 20 the first stage, the number of cells discharged will ooo,: exceed the number of cells newly formed. On the other hand, at a rate of flow which its too low, cell growth 0 a S°oo might cease in certain circumstances.

Such a process has the disadvantage that, on the one hand, the productivities scored are not satisfactory, and on the other hand, long-term stability of the f *continuous culture is not established, this implies that over a protracted period of one-hundred hours and more the rate of production cannot be kept constant. This is due to the products being formed, viz. butanol, acetone and/or ethanol, possibly also isopropanol, having a toxic effect on the fermenting organisms, so that at a certain concentration stage of the products being formed either the formation of these products is halted or the bacteria -oi!s w K.0 2.4^ -2-

I

4444r 4i1 tf( 4r 4 14444 cultures are severely damaged. A further disadvantage of this known process may be considered to lie in the fact that moderately satisfactory results can only be obtained with hexoses.

The aim of the present invention is to create a process of the kind described further above, by means of which consistently favourable production figures can be obtained over a lengthy period of time. In addition, the process has to make feasible the use of source materials that do not contain hexoses exclusively.

This problem is solved according to the invention by having the bacteria immobilised on a carrier in the second stage.

For single-stage processes, means for immobilising 15 bacteria are already known, inasmuch as in such processes cells are immobilised in Ca-alginate and glucose is applied as the carrier. In these familiar processes productivity figures could in fact be increased, but only low productivity could be maintained over a lengthy fermentation period, e.g. over several weeks.

The process proposed by the present invention is very flexible as to the rate of flow, since the viable bacteria will cling to the carrier material in the second stage by adhesion and thus the rate of flow can be so adjusted in the first stage of the process that the first stage is almost exclusively restricted to the production of bacteria, a portion of which is then carried along into the second stage of the process. In this second stage, then, a large population of bacteria is obtained, since the viable bacteria cling to the substrate material, whereas the dead bacterial cells are flushed out with the medium enriched with the product. One can, therefore, in the first stage operate with a rate of flow that is sufficiently high to preclude the products formed from exceeding the limit of toxicity; thus the bacterial culture will remain substantially undamaged. It is expedient to use open-pore sintered glass, or pumice 44 4 4 4 1r

I

s r: i u r i i i FRA42, -13$

N

i Y I i -3 stone, or an equivalent substance as carrier material, such substance having a preferably high Si02-content, preferred pore-sizes between 20 to 200pm, more preferably 50 to lOO1pm and a preferred water-absorbing capability or equal to, or greater than 0.35ml/ccm, while the ratio of the volume of the carrier material to the total process volume is preferably between 1:10 and 1:1.1, more preferably between 1;5 and 1;6, resulting in a particularly effective fermentation, especially if: in the first process stage, a preferred rate o lo of flow of 0.08 to 0.45/h, but preferably 0.1 alto 0.38/h, a pH of 4.0 to 5.5, but preferably 4.3 to 5.1 more preferably 4.5 to 5.0 and/or a temperature of 30 0 C to 40 0 C, but preferably o o 15 33 0 C to 38°C; and 1Oo in the second progress stage. a preferred rate of flow of 0.14 to 0.4/h, but preferably 0.25 to 0.35/h a pH of 3.0 to 5.0 preferably to 4.8 but preferably 4.2 to 4.5 and/or a 20 temperature of 27 0 C to 40 0 C, particularly 000 to 37 0 C but preferably 32 0 C to 35 0

C.

are maintained, the reactor content having been thoroughly mixed during the first stage.

o.ao.. In a further preferred implementation of the process proposed by this invention, a quantity of medium goe is drawn off in the product-formation stage, the product formed is separated by extraction, preferably in the commonly-known manner by means of higher alcohols or higher fatty acids and the phase thus obtained, which is i low in product, is recycled into the product-forming stage and/or into the first process stage. In this manner, product concentration in the second stage is kept low, thus preventing damage being sustained by the bacteria, so that a very high product-formation rate is maintained always, even over a very long period of time Furthermore, in this way it becomes feasible to carry out i the separation action without affecting the fermentation j- Ii i

II(

I I r -4stage proper. In the second stage, however, product may be drawn off from the medium by pervapocation, preferably in a continuous manner. In so doing, a pervaporation diaphragm, preferably made of silicone rubber, can be used for the pervaporative removal of the product, while maintaining a temperature of 30 to 90 0 C, preferably 40 to 0 C and while bringing about a reduction in the pressure on the pervaporation side by means of a carrier gas, such as e.g. N 2

H

2

CO

2 or by mixtures of these gases, or else by a fermentation gas or by applying a vacuum, respectively. In the second process stage, however, the separation of the product can also be performed by means of gas stripping, preferably continuously. For such continuous separation of product by gas stripping, e.g.

N

2 H2, CO 2 or a mixture of these gases, or else fermentation gas may be used, the volumetric ratio of liquid flow to gas flow being in the range 0.05;1 to 1.6:1 and the temperature in the range of 30 0 C to 90 0

C,

preferably 40 0 C to 80 0

C.

All the processes for the separation of product outlined above result in an increased productivity, in the possibility to utilise carbohydrate solutions of higher concentration, and in a greater long-term stability, as well as in the possibility to utilise unconventional types of sugar.

In order to allow for an especially accurate control in the running of the process, the feed rate of nutrient solution and/or sugar solution can be controlled in the second stage as a function of product content. In this manner, the settings can be made to obtain precisely those concentrations of nutrient solution and of sugar, respectively, which are required for the optimal running of the process.

For the implementation of the process proposed by this invention an apparatus based on the same invention is proposed, consisting of a bio-reactor, preferably an agitator-vessel bioreactor; and of a reactor equipped with material that will retain micro-organisms, preferably a fixed-bed reactor, both connected in series, with the fixed-bed reactor having an external loop connected to it for the, preferably continuous, separation of product. Such a combination of reactors allows in a particularly simple manner for the control of the rate-of-growth of micro-organisms employed on the one hand, and the formation of product on the other, in the second stage of the process.

In this arrangement, one or more feed-lines may be Ott provided within the fixed-bed reactor in order to enhance ,circulation in that reactor. In this manner, an approximately uniform distribution of nutrient carrier and also of product within the reactor is obtained. In particular, circulation may be facilitated by local gas-feed devices or conduits for the direct inflow of, preferably recycled, medium. When gas is being introduced, circulation is achieved by air-lift pump action, whereas in the case of directed entry of liquid S 20 the suitable rate of flow will facilitate circulation.

A pervaporation module can be inserted in the external loop, for product separation. Within the pervaporation module, a diaphragm, preferably made of silicone rubber, may be provided. This will result in a particularly favourable separation of the product from the medium by directing the flow in cross-current past the diaphragm, a possible sedimentation of micro-organisms on the diaphragm may be prevented.

Finally, a counter-current stripping column may be inserted in the external loop for the separation of product. The choice of the alternative for product separation will then depend on the material at the outset, on the bacteria used and also on the product being formed.

In summing up, it is to be noted that in the first stage of the process a sufficiently high rate of flow is chosen to keep the bacteria in their growth-phase and L L their acid-forming phase, without any product formation worth mentioning. In this growth phase the bacteria t develop good adhesive properties which, on their transition into the second process stage, result in the immobilisation of bacteria upon the carrier material.

Should rate of flow be selected in the first process stage which is too low, then cell growth, and possibly also product formation, are stopped and thus the goodI absorption properties of the bacteria with respect to the carrier material are lost.

Immiobilisation in the second process stage may be replaced by separation of cells by micro-itaon preferably cross-flow microfiltration from the medium and.

subsequently by their recycling; this will have the same effect as immobilisation.

P In addition, too high a product concentration in the first stage (at a low rate of flow) will result in a degeneration of the bacteria, which in turn will cause a change from product formation to a renewed formation of acid, and experience shows ;jj 6 I that this change is irreversible.On the other hand, a too high flow rate in the first stage might cause the bacteria to be flushed out, so that after some time the supply of bacteria for the second process stage becomes insufficient.

Therefore, the rate of flow has to be adjusted in each individual case according to the prevailing conditions.

In the drawing, various examples of application of equipment based on the present invention are shown schematically. Fig.l shows the schematic diagram of the process; here the product formed is separated by pervaporation. Fig.2 shows the schematic diagram of a process in which separation is effected by product extraction. Fig.3 shows the schematic diagram of a process in which the product is expelled from the medium by stripping.

In all three application examples, corresponding parts have been labelled by the same symbols. A storage veesel is denoted by 1; out of this, the nutrient solution is fed via a line 2 by means of a pump 3 into the first process stage, formed by an agitator-vessel bio-reactor 4. The nutrient substrate enriched with bio-mass is fed from the vessel reactor via a a line 5 by the pump 6 to the second process stage,:which is formed by a fixed-bed reactor 7. From this fixed-bed reactor, a line 8 leads to pump 9, by means of which the medium enriched with product is discharged from the fixed-bed reactor..

In the application example according to Fig.l, the medium enriched with product is passed via a heat-exchanger which is heated by the back-flow of medium already freed from product. The hot medium, rich in product, is passed to a heater 12 via the line 11; from there,it is passed to a pervaporation module 13.This pervaporation module 13 contains a diaphragm 14,with medium on one of its sides and carrier gas on the other.Through this membrane,a product exchange is taking place,inasmuch as gas is passed via line 15 into the pervaporation module 13. The product-free medium is passed via the line 16 and the pump 17 to the heat exchanger 10 and is then recycled again via the line 18 into the fixed-bed reactor.

Excess medium to a great extent free of product,is discharged Io 1 i -0 7 via the line 19 A portion of the medium ,as shown in broken lines in Fig.l,is fed via line 20 by a pump 21 to the agitator vessel bido-reactor. The gas enriched with product is fed from the pervaporation module 13 via line 22 to a cooling device 23, in which the product formed is condensed and is drawn off via the line 24. The gas freed from product is recycled to the pervaporation module 13 via the line In the practical example according to Fig.2, the medium drawn off the fixed-bed reactor, enrichei with product, isi' fed via line 8 by the pump 9 to a heater 30,from where it enters a mixer 31. An extraction agent is fed into this mixer via line 32 and the mixture of extraction agent and of product-enriched medium is fed into a separator 33 via line 34. In the separator 33 the product-carrying extraction agent and the product-free medium are separated from each other, the extraction agent enriched with product being drawn off via the line 35 and the product-free medium via the line 36. The medium freed from product is then recycled into the fixed-bed reactor via line 37; excess medium is drawn off via line 38. As indicated in broken lines in Fig.2, medium freed from product may also be recycled into the first process stage by a pump 40 via a line 39. The separation of extraction agent and product may be carried out by conventional means, by distillation or the like.

In the application example according to Fig.3, the medium enriched with product is drawn off by pump. 9 over line 8 and is subsequently fed to a heat exchanger 10,from which it is passed to a heater 42 via a line 41 and from where it enters a stripping column 44 via a line 43. In this strippirg 4, column the medium,enriched with product, is treated in counter-current with gas,the latter being injected into the column by pump 46 via line 45. The stripping gas, now enriched with product, is fed via line 47 to a cooling device 48, wherein the products are condensed and are drawn off via line 49.The gas freed from product is again recycled SP L t 1 -8 to the stripping column 44 by the pump 46 via the line 47. The medium freed from product is fed to the heat exchanger 10 by the pump 51 via the line 50; in the heat exchanger the medium, still warm, encounters medium coming from the fixed-bed reactor 7. From the heat exchanger 10 the medium freed from product passes via line 52 into the fixed-bed reactor 7. Again in:;this case, as before, excess medium is drawn off the system via a line, 53.The line 54, shown in broken lines, and the pump 55 again serve the purpose of recycling the medium freed from product into the first process stage, viz. into the agitator-vessel bio-reactor 4.

The process proposed by the invention shall be illustrated below in greater detail by means of concrete examples.

Example 1: A medium having the composition as stated below is used as nutrient medium: Xylose (anhydrous)

KH

2

PO

4 Ig/l

K

2

HPO

4 1g/l

(NH

4 2 S0 4 2g/i MgSO,7H20 0.lg/l NaC 0 001g 1 Na2Mo04.2H20 0.01g/l CaC12 0.01g/l MnSO 4

H

2 0 0.015g/l FeSO 4 7H 2 0 0.015g/l Biotin 0.1g/l Thiaminium chloride 0.002g/l t p-aminobenzoic acid 0.002g/l Na2S 2 0 4 0.035g/l Resazurin, 0.001g/l Yeast extract Na OOCCH 2.2g/l Clostridium acetocutylicum ATCC 824 is applied as the fermentation organism, grown in a suitable preliminary culture prior to its intxrouction into the process proposed by the present invention.

aR 1

I

(NH^SO^ 2g/l I, -9- The nutrient medium issuing from the storage tank 1 is fed into the agitator-vessel bio-reactor 4, specifically at a rate of flow of 0.3/h, the inoculum being approximately 10% of the volume of the agitator-vessel bio-reactor 4. Fermentation is carried out in the agitator-vessel bio-reactor at a temperature of 37 0 C, while a pH of 5.1 is being maintained inside the reactor. In view of the rate of flow chosen, cell density inside bio-reactor 4 is beinq kept fairly uniform and the new cells arising through the growth of bacteria S, are fed via line 5 into the fixed-bed reactor 7, where they become attached to the carrier material by adhesion. Open-pore sintered-glass is used as carrier material, the volume the sintered-glass material amounting to 50% of the total process volume. In the fixed-bed reactor 7 a fermentation temperature of 330C is maintained, the fermentation being carried out at a pH of 4.3. The total rate of flow after both process stages have been passed is 0.15/h. Within the fixed-bed reactor 7, the contents of that reactor are treated with nitrogen gas and in view of the directional flow devices present in the fixed-bed reactor, circulation will ensue.

Productivity and yield are compiled in Table 1, below: TABLE 1 (g h (g h) i) (mV) S 0.15/h (race of flow).

I: '0 04 D 0.15/ (rate of flow).

10 Example 2: The same nutrient composition, the same inoculum and the same process conditions are used as before within the agitatorvessel reactor and the fixed-bed reactor. An external loop is connected to the fixed-bed reactor; through this the products formed can be drawn off continuously or in batches. To this end, the liquid drawn off the fixed-bed reactor,which is enriched with product, is heated to a temperature of approximately 40 C and fed to a stripping column in which nitrogen is introduced as a stripping gas, in counter-current to the liquid.

The volumetric ratio of liquid flow and gas flow is 0.35 1.

The liquid medium, largely freed from products and cooled to fermentation temperature, is partly recycled into the fixedbed reactor 7, while a smaller portion is recycled into the agitator-vessel bio-reactor 4.

The excess liquid, largely freed from product, is drawn off from the system. The gas drawn off the stripping column, enriched with product, is passed over a cooling device,in which the products can be cooled and drawn off. The steadystate values obtained are summarized in Table 2.

TABLE 2.

00 o 0 0 0 36 4 H 1 D b l I HH 4 e o I )3 o o 0 0d H H o H o H 4d 0 *ri n d r W Cd d bO W) 4P H-1 H P (mV) t 21.'0Q p2.50l5.376.82 10.87 1.638 .76 6.59 4.10 -450 130.3 41 D 0.15/h (rate of flow) -C C-- Lti

V-

11 As may be seen from the Tables, productivity and sugar degradation in the process according to Example 2, with continuous removal of product through an external loop, are significantly better. The same conclusion results also,as shown by the tests above, when fermentation is carried out over a longer period of time, in the case in point over 500 hours.

I tl" iI

Claims

1. A process for the fermentation of media containing carbohydrates by the use of bacteria forming butanol, acetone, ethanol and/or isopropanol, said process being carried out in at least two stages, of which in the first stage bacteria are being cultivated substantially continuously, and in the second stage the product is being substantially formed, either continuously or in batches; characterised by the bacteria being immobilised in the second stage, of the process on a carrier material.

2. A process according to Claim 1, characterised by open-pore sintered glass, pumice or an equivalent substance being used as carrier material such substance having high SiO 2 -content, an open-pore structure, a pore-size of 20 to 200pm and a water-absorption capability of at least 0.35ml/ccm, while the volumetric ratio of carrier material to total process volume is in the range of 1:10 to 1:1.1.

3. A process according to Claim 1 or 2, characterised by medium being drawn off in the product-forming stage, the product formed being separated by extraction; and the phase thus obtained, low in product, being recycled into the product-formation stage and/or into the first process stage.

4. A process according to Claim 1 or 2, characterised by product being drawn off by pervaporation during the second stage. A process according to Claim 4, characterised by a pervaporation diaphragm, specifically one made of silicone rubber, being used for drawing off product by pervaporation, while temperature is maintained between 30*C to 90*C and while a reduction in vapour pressure is attained on the pervaporation side by a carrier gas such as N 2 H 2 CO2, or by a mixture of these gases, or by fermentation gases, or else by the application of a vacuum. i L i 7 perapraio diprgsecfclyoe aeo rub r ben sd f rd ain f rd c y c o n c e n t r a t i o n c Y u the formation of these products is halted or the bacteria RA4/- 13

6. A process acco-ding to Claims 1 through 3, characterised by product separation by means of gas stripping taking place, by means of stripping with gas, in the second stage.

7. A process according to Claim 6, characterised by a stripping gas, or else fermentation gas being used for the continuous separation of product by gas stripping, whereas the volumetric ratio of liquid flow to gas flow is between 0.05: to 1.6:1 and the temperature is between to

8. A process according to Claims 1 through 4, characterised by the rate of nutrient solution and/or sugar solution being supplied is being controlled as a function of product content in the second process stage.

9. An apparatus for carrying out the process according to any one of the Claims 1 through 9, characterised by a bio-reactor, and a reactor equipped with substances capable of retaining micro-organisms, being connected in series, while the facility exists to connect an external loop in the fixed-bed reactor for the separation of product. An apparatus according to Claim 9, characterised by one or several devices controlling the direction of flow being installed within the reactor, in order to facilitate circulation taking place.

11. An apparatus according to Claim 10, characterised by local gas feed devices or conduits for the directionally controlled inflow of media being used, in order to facilitate circulation.

12. An apparatus according to Claim 9, characterised by a pervaporation module being inserted into the external loop for the separation of product.

13. An apparatus according to Claim 12, characterised by a diaphragm, specifically a diaphragm made of silicone rubber, being contained in the pervaporation module. I i 'l-o r o culture will remain substantially undamaged. It is expedient to use open-pore sintered glass, or pumice 14

14. An apparatus according to Claim 9, characterised by a counter-current stripping column for the separation of product being placed in the external loop. A process according to Claim 2, wherein the pore-size of the open-pore' structure is 50 to 100pm.

16. A process according to Claims 2 or 15 wherein the volumetric ratio of carrier material to total process volume is in the range of 1:5 to 1:1.6.

17. A process according to Claim 3, wherein the product formed is separated by extraction in the generally-known manner by means of higher alcohols or of higher fatty acids.

18. A process according to Claim 4, wherein the product is drawn off continuously.

19. A process according to Claim 5, wherein the Stemperature is maintained between 40 0 C to 80 0 C. A process according to Claim 6, wherein the gas stripping is continuous.

21. A process according to claim 7, wherein the temperature is between 40 0 C to 80 0 C.

22. A process according to Claim 7 and 21, wherein the stripping gas is N2 H CO 2 or a mixture.

23. An apparatus according to Claim 9 wherein the bio-reactor is an agitator-vessel bio-reactor.

24. An apparatus according to Claims 9 and 22 wherein the reactor is a fixed-bed reactor. An apparatus according to Claims 9, 22 and 23 wherein separation of the product is continuous.

26. An apparatus according to Claim 11 wherein the media is recycled. DATED this 6th Day of NOVEMBER, 1991. VOGELBUSCH GESELLSCHAFT M.B.H. Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS 1 1 A. uV T 0 i the separation action without affecting the fermentation 15 Summary The fermentation process of media containing carbohydrates with the aid of bacteria forming butanol, acetone, ethanol and/or isopropanol is carried out in at least two stages; essentially, bacteria are being substantially continuously cultivated in the first process stage and product being formed in the second process stage, continuously or in batches. In order to attain a long-term stability of the process, to increase the productivity of the process and to enhance sugar degradation, the bacteria are immobilised on a substrate material in the second process stage. In so doing, medium may be drawn off from the product-formation stage and the product formed may be separated, to prevent inhibition occurring due to excessive concentration of product. In an apparatus for carrying out this process a bio-reactor, preferably an agitator-vessel bio-reactor and a reactor equipped with substances retaining micro-organisms,preferably a fixed-bed reactor are connected in series; where indicated,an external loop may be connected to the fixed-bed reactor in order to facilitate the,preferably continuous, separation of product. The:, femnainpoeso eiacnann abhdae