DE2633076A1 - METHOD OF CARRYING OUT ENZYMATIC REACTIONS USING MICRO-ORGANISMS INCLUDED IN A POLYMERIZATION MATRIX - Google Patents

Description

PATENTANWÄLTE A. 3RÜNECKER

W. STOCKMAIR

K. SCHUMANN

DR REH NAT. ■ DIPL-PHYa

P. H. JAKOB

CMPL-tNG.

G. BEZOLD

DR BER NAT · DIPL-CHEM.

8 MUNICH

MAXIMILIANSTRASSE

July 22, 1976 P 10 658 _60

INSTITUT NATIONAL DE LA EEGHERGHE AGRONOMIQUE (INRA) Rue de Grenelle
Paris 7i France

Process for carrying out enzymatic reactions using microorganisms enclosed in a polymer matrix

The invention "relates to a method for carrying out enzymatic reactions using in
microorganisms enclosed in a polymer matrix,

The importance of enzymatic Eeaktionen in the technical field does not need to be more feetont _s because virtually

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ORIGINAL

there is no longer any example of a field of organic chemistry in which enzymatic reactions are not present be performed. Carrying out these reactions, however, poses certain problems that have hitherto been encountered have not been completely satisfactorily resolved. The following are the basic ways of performing the enzymatic Reactions are summarized without giving a precise example, as these are very are numerous and well known.

The first way of carrying out enzymatic reactions consists in isolating the enzymes from microorganisms and then using them as simple catalysts either in free form or enclosed in polymer matrices. This Verfahren.sind generally very complicated, because in the majority of these are the isolation of the enzyme requires a complicated technique _o, It is to methods intended for small scale syntheses of products, such as drugs, reserved.

Another way of performing enzymatic reactions, especially for the treatment of effluents, is used, consists in that the bacteria in a solution containing the compounds to be broken down, grows, e.g. the milk serum from cheese factories, which also contains the elements of a nutrient medium; that is what it is fermentation (fermentation) in the non-stationary State, in this case one can maintain a relatively constant activity because of the dead microorganisms are continuously replaced. But besides the problems with the cultivation of free microorganisms that lead to the general

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AL INSPECTED

is forced to use discontinuous processes, growth often enough is also a disadvantage the total enzymatic activity of the reaction. So is the yield of citric acid fermentation of Aspergillus niger due to the absence of nitrogen as well as full other elements of the milieu.

For this reason, enzymatic reactions with free microorganisms are used in certain ways of carrying out enzymatic reactions preferably a solution containing the product to be treated and a "poor" nutrient medium as the reaction medium or no nutrient medium to slow down the growth of the microorganisms and to promote their enzymatic ^ activity, this is a fermentation in the steady state ^ one finds, however, that the enzymatic activity decreases fairly quickly over time

In both of these embodiments, the main disadvantage is undoubtedly the fact that the microorganisms, particularly the bacteria, represent products which are difficult to handle and which can not form action columns, the fillings in the R _e, in particular, "

In order to eliminate this disadvantage, fillings have been developed that consist of microorganisms that are attached to a Carriers are adsorbed. However, experience has shown that these are non-stationary cultures, which still suffer from certain of the disadvantages mentioned above. It must also be noted that this The type of filling, the activity of which is localized on the surface, does not have an equally large coefficient of effectiveness like a filling, the activity of which is distributed over the entire volume.

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Recent research in this area have hand Haberen to the development of better and more active microorganisms by inclusion of the same in Polymerisatmatrizen out, for example, by polymerization of a monomer and the microorganism solution containing · This industry made Polymerisatkörnchen available, a similar enzymatic activity as the have trapped microorganisms. Heretofore, these polymer granules have been regarded almost as much as the enzymes blocked in the resins described above which are also in a more economical form, assuming that the activity of the entrapped microorganisms is generally lower than that of the blocked enzymes. This is because it was assumed that the inclusion of the microorganism in the polymer matrix prevents the latter from developing and only offers the advantage that the enzymes it contains can be used particularly conveniently. Therefore, in the continuous reactions in which polymers containing blocked microorganisms are used, the reactor is supplied with a solution which contains only the product to be treated. In general, a decrease in the activity of the polymer granules can be observed at the end of a certain period of time, so that one is forced to renew the filling of the reactor.

However, it has now surprisingly been found that it is possible to enzymatic R _e perform actions using a polymer matrix enclosed in micro-organisms, without the enzymatic activity "decreases the same time.

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The aim of the invention is to develop a method for carrying out enzymatic reactions in the disadvantages outlined above do not occur.

The invention is a method for carrying out enzymatic reactions in which in a Polymer matrix enclosed microorganisms are used, which is characterized in that one in a reactor which contains the microorganisms enclosed in a polymer matrix, continuously Introduces nutrient medium for the microorganisms, which contains the (s) to be treated (ή) product (s), and that one the reaction products are continuously withdrawn, ensuring that the trapped microorganisms in remain in the reactor.

This process is particularly interesting (advantageous) when it is carried out continuously, ie because a reactor is continuously charged, which can have any shape, for example that of a filled column or that of a reactor with an overflow, with a nutrient medium for the microorganisms containing (s) to be treated (s) product (s) and the continuously withdrawing the reaction products in which wherein it is ensured that the entrapped microorganisms in the R _e remain actuator.

The method according to the invention offers numerous advantages. On the one hand, they can be enclosed in a polymer matrix Microorganisms easily handled and exemplified

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wisely be introduced into the reactor as a filling, on the other hand As far as we know, the enclosed microorganisms are subject to practically no growth and thereby the total enzymatic activity of the Milieus are not changed, as is the case when free microorganisms are cultivated on a nutrient medium. However, there is also no reduction in the enzymatic activity, as is the case when a poor culture medium is used is to be observed. This phenomenon, which is surprising in itself, can be stated, for example, as follows are explained, but the invention is by no means based on the theoretical explanation given below is limited.

The microorganisms enclosed in a polymer matrix are not in because of their steric separation able to multiply despite being supplied with a nutrient medium, but it has been found that these trapped microorganisms a very considerable Degree of renewal of their proteins ("turn-over") show when they are supplied with the nutrient medium, which the Maintaining enzymatic activity could explain this. Obviously, this can renew the proteins do not take place in a poor nutrient medium for free bacteria in the steady state, because from this environment voluntarily Certain elements have been removed that affect the growth of microorganisms and the synthesis of proteins favor.

According to a preferred embodiment of the invention Process is the polymer used a polyacrylamide. It is also possible to perform this procedure using any of those known in the art Methods such as those of Hicks and Updike (1966)> to be carried out. Of course, others can too

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Polymers are used as polyacrylamide, provided that they can include microorganisms, without them being destroyed in the process, and provided that these microorganisms also have their enzymatic properties Activity (effect) can exert on a solution outside of the polymer.

The solution that is introduced into the Re.aktor and the the product to be treated contains also generally contains, as is known per se in the field of biology, a source of assimilable carbon, such as carbohydrates, e.g. glucose, fructose, lactose, maltose, Sucrose and pentose, molasses (sugar syrup), starch and usable carboxylic acids, a source of assimilable nitrogen, such as organic nitrogen compounds, such as peptone, brewer's yeast extracts (yeast extracts), meat extracts, amino acids, or inorganic nitrogen compounds, such as an ammonium salt, as well as a source of inorganic salts such as potassium monophosphate, sodium acetate and the like. This solution can also contain other additives required for the growth of the microorganisms such as vitamins, oligo elements and growth factors. Of course, in certain Cases the product to be treated also serve as a carbon source, nitrogen source and the like or also certain, contain elements necessary for growth.

The other parameters of the reaction, such as the pH value and the temperature, are generally adjusted so that like an IT medium for the growth of microorganisms common·

In one of the embodiments of the method according to the invention the trapped microorganism is a bacterium of the genus Lactobacillus, in particular

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others around Lactobacillus casei or Lactobacillus delbruckii, which allows the production of lactic acid from malic acid or from sugar

In another embodiment of the method according to the invention if a bacterium of the genus Pseudomonas, in particular Pseudomonas aeruginosa, is used as the microorganism, to carry out denitrification; for this purpose the solution to be treated, which contains nitrates, is added Compounds that allow this bacterium to grow, especially a carbon source.

In a further embodiment of the invention In this process, a yeast fungus is used as an enclosed microorganism to carry out the alcoholic fermentation of the genus Saccharomyces or Schizosaccharomyces, in particular Saccharomyces cerevisiae or Schizosaccharomyces pombe In this case, a substance is fed into the reactor that contains sugar, such as molasses (sugar syrups), Wine must (mout) or fruit juices. In the case of molasses, which are not a sufficient nutrient medium, one must still add a nitrogen source such as ammonium sulfate.

The preferred embodiments of the method according to the invention described above are particularly advantageous, The method of the invention can also be used to carry out other enzymatic reactions, where bacteria, yeasts and molds can be used.

The method according to the invention is particularly applicable to carry out enzymatic reactions in which mutants of microorganisms are used, especially represent advantageous enzymatic starting materials, which on the other hand also generally themselves

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Rather little growth on rich culture media and yet deliver particularly advantageous results under the conditions according to the invention.

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example 1

Production of the microorganisms enclosed in a polyacrylamide gel ■ __

The gels are made according to that described by Hicks and Updike (1966) Process made. The microorganism is grown on a growth medium (growing medium) (hereinafter referred to as "rich milieu"), which leads to treating product contains to initiate the synthesis of the corresponding enzyme. The cells are subsequently washed in a phosphate buffer with a pH of 7 and centrifuged.

Thereafter, the above-mentioned polymerization is carried out as follows: the microorganism is suspended in a 0.05 M phosphate buffer (pH 7) at a temperature of 15 C to form a suspension which, for 100 ml of solution, 8.2 g of the monomers acrylamide and Contains ΪΤ, ϊΤ-methylenebisacrylamide (81 % acrylamide) and as a catalyst 200 mg ammonium persulfate and 40 microliters of N, N, F ¹ , N ¹ -tetramethylene diamine. The polymerization is carried out in such a way that the solution is kept at a temperature of 15 ° C. for 1 to 2 minutes while passing in nitrogen.

The gel obtained in this way is thoroughly rinsed with an aqueous solution, then into particles a few mm in size comminuted, which represent the filling of the reactors described in the following examples.

Example 2 Conversion of L-malic acid into L-lactic acid

The reactor used in this experiment is a flask with a tissue culture (Celstir),

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from which the reaction products overflow through a filter covered with a nylon membrane, which retains the particles in the medium will. The medium is stirred using a magnetic bar floating in it, which prevents that the .Polymerisatkornchen lying on the bottom of the reactor are pulverized.

The microorganism used in this process is the strain Lactobacillus casei, deposited, in the Collection de L-IMRA de Montpellier under the No. CAP ^ C ^ i. These bacteria are on a culture medium bred, which has the following composition:

Brewer's yeast extract (Difco) 4- g

Tryptone (Difco) 2 g

KH ₂ PO ₄ 2 g

Mineral salt solution according to Skeegs and Wright * 5 ml

clarified tomato juice 80 ml

Glucose 10 g

DIr-malic acid 8 g

Water ad 1 1 pH adjusted to 4.7 with 10 η IaOH

* MgSO ₄ .7H ₂ O: 40 g / l; MnS0 ₄ .4H ₂ 0: 2 g / l; PeCl ₅ : 0.4 g / l and pure HCl: 1 ml / l

which has been sterilized ^{at 110 ° C. for 20 minutes.} 1 liter of this medium gives 950 mg of bacteria (based on the dry weight) after 48 hours, which can be used for the production of 50 mg of polymer particles, as described in Example 1.

A medium (A) is introduced into the reactor which has the following composition:

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Brewer's yeast extract (Mfco) Tryptone (DIfco) Tomato juice

4th S. 5 2 G 80 ml 2
1 I. G ¹ *
8th S. 5 ml 90 ml 50 mg ad 1 1 3,

glucose
DL maloleic acid

Mineral salt solution according to Skeegs and Wright 95% ige3lthanol
Cycloheximidine
'Water
PH value . ■

This is a culture medium of the type described above, which contains the compound to be treated,. ie DL-malic acid, and also contains cycloheximidine and ethanol, the latter two compounds being added in order to avoid contamination by yeasts and other bacteria. At the degree of dilution used in this experiment, no development of free lactic acid bacteria can be observed, so that the conversion of the malic acid took place solely through the fixed bacteria. The decarboxylation of malic acid to lactic acid is carried out after the manometric method by Warburg under a nitrogen atmosphere at a temperature of 30 ⁰ S; this is the following basic reaction:

0
HO - OH - C - OH + enzyme> HO - CH - C - OH + CO ₀

j I It ^

CH ₀ -C-OH ^CH 3 ° "

The 'enzymatic dosages of L-lactic acid and the L-malic acid is determined according to the methods of Barre (1966) and Poux (1969) proposed method performed using pig muscle meat dehydrogenase malate

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INSPECTED

(commercially available from Boehringer) * Die enzymatically ^ Dosing of the glucose is according to the method carried out by Keilin et al (194-8) after Staub (1963) .

At the same time, experiments are carried out in which nutrient media the following composition can be used:

Environment (B) tomato juice

₂₄ glucose. DIr malic acid

Mineral salt solution according to Skeegs and Wright 95% ethanol
Cycloheximidine
water
PH value

milieu

10 ml 5 2 S. β 1 S. G 8th G β 5 ml ml 90 ml ml 50 mg mg ad 1 1 1 1 3, 5 2 -1 8th 5 90 50 ad 3,

Glucose Dlr-Malic Acid

Mineral salt solution according to Skeegs and Fright

95% ethanol

Cycloheximidine

water

PH value .

The useful volume of the reactor used is 150 ml (volume of liquid + volume of particles) and the Feed rate is 38 ml / hour.

The results obtained are in the attached Drawing shown graphically showing the course of the

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L-malic acid concentration in the effluent as a function reflecting time. It should be noted that when carrying out the method according to the invention with the Environment (A) the activity remains constant or even increases, while when using the environment (C) after 5 days. and when using the milieu (B) after 15 days the activity has practically decreased to 0.

The observed increase in activity in the process of the invention is very general. Thus, in the lactic acid conversion, after 9 months of continuous use of the reactor, the activity of the particles is more than five times higher than. it was at the beginning of the experiment. This increase in activity is determined by isolating the particles contained in 1 ml of solution and testing their activity against a lactic acid solution: on 11/21/1973 this activity was 130 nl CO ₂ / ml. Hours - on June 13, 1974 - this activity was 750 / al GO ₂ ZmI . Std. This is, of course, what is released during the reaction

The increase in activity can be explained by the fact that the nutrient medium promotes the conversions in one direction stigt, which is always favorable to the reaction. This is a very interesting advantage of the method according to the invention.

Example 3 Conversion of L-malic acid into L-lactic acid

In a similar reactor as in Example 2 is a solution (A) - (see Example 2) in a feed rate of 38 ml / hour. introduced. This reactor has been running for 10 months with a constant productivity of 14.4 mmol / 1. Hours. and allows the conversion of 1.9 g / l · hr. L-malic acid.

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The degree of ^ P ^ odukijivity is calculated as follows: m _ Q 2

C = Proiuktfconzentrat ion im. the drain for that. _; .-Sya $ heseil or, the difference between the- _: sample concentration in the charge and: 4 _; Product concentration, in atomic stream during degradation;

D = levy amount ;;

ΐν- · βξ "'Volume of the liquid at the same time

By VerÄoppiüng ^ OCE - ZellJton-zen ^ r ^ _'ioiL: - bÄi organization of the Pjoly': Geis ^ can this], special J Produktivität'des, Eeaktor ^ ^ In double _in;. : the ^ e. ^ e_ise has succeeded, starting from the same; Ifpjsujpg _; -w4..e pbenjj ■;: ■; ■■ .-. by increasing the amount of. in,; the gel: inculcated bacteria-s pro-, 'Litei * -undiStuäide ^ 7-Ig ₁ -Ajfcf el ^ acid. ^ to convert. ■

This type of reaction is particularly interesting (advantageous) for the »application: 'bei'-der: \ , W: ei.nhiöri £ it, ell ^ igs -iZja |? .. [^ creation ^,:; - ■;

Example: M- - · ^; · ■; ': -i ■; -.- · ■ _ ■ :: j \. ■■ -. · -. -.-: - ■ 2-5 i: _ ^ - ■■ - ■ "■ --.- · - ■ _ ,, · · £ .- - - ■ _-:: denitrification by Pseudomonas aeruginosa -;?. ,,

The bacterium is in one. Nutrient Broth "), cultivated, which contains 500 mg / 1 potassium nitrate. After 3 days, an Ifoltimön of, g I ₁ - the; · -, <lg bacteria, based on the dry weight -,:; represents _.: - _: , : ■■ · ■ -.;

These. Bacteria are 'subsequently in a Pplymerisat * - matrixnacfc> the one described in, Example / 1: ^ Experienced e _v d

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closed to obtain 50 ml of particles. To carry out the reaction, a reactor of the type described in Example 2, which has a volume of 150 ml and to which an environment is used, is used
supplies the following composition:

Potassium nitrate 2 g

Glucose 2 g

Monosodium phosphate 5 g

Mineral salt solution according to Skeeks and Wright 5 ml

Oligo element solution (1) 1 ml

Water ad 11 pH 6.5, adjusted with 10 η NaOH

(Ό Oligo element solution_:

Potassium molybdate 0.05 g

Sodium borate -. '0.05 g

lerriferrochloride 1 drop

Eobalt nitrate, 0.05 g

Cadmium sulfate "0.05 g

Copper sulfate 0.05 g

Zinc sulfate - 0.05 g

Manganese sulfate 0.05 g

distilled water ad 1 1

The feed rate is 48 ml / hour. The following results are obtained:

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Days nitrate content in the effluent (s / l) 1st day 1.03

3rd day 0.98

4th day 0.68

7th day

15th day 0

From the above results it can be seen that from the 7th day the denitrification of the solution is complete.

example 5

Alcoholic fermentation of grape juice

The strain used in this experiment is the yeast Saccharomyces cerevisiae, which is found in the alcoholic fermentation of wine isolated and in the I.N.R.A. has been deposited by Dijon under the number STV III.

The nutrient medium for the yeast is a Wickerham malt containing 50 g / l glucose. To Stands, the yeast fungi originating from 1 liter of this milieu are obtained for the production of 70 ml of polymer parts according to the procedure given in Example 1.

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The fermenter consists of two reactors of the type described in Example 2 in cascade form, each having a capacity of 250 ml. White grape juice with a pH value of 3 _t 1 and a sugar content of 126 g / l in a quantity of 80 ml / hour is fed into the fermenter. a.

Each 1-month operation of the reactor is obtained

- in the reactor 1 0.40 mmol / ml ethanol,

- in the reactor 2 0.85 mmol / ml of ethanol.

The productivity guard of these reactors is thus:

- T = 180 mmol / 1 χ hours for reactor 1 and

- T β 210 mmol / 1 χ hour for reactor 2.

The fermenter allows the production of 18 g of ethanol per liter and hour.

Comparative tests carried out using free cells in normal fermentation have shown that the optimum level of productivity is 16 mmol of ethanol per hour and hour. It should be noted that the normal fermentation proceeds at 3O ⁰ C, while the fermentation according to the invention at 25 ° C is performed, thereby the productivity can be further improved. In addition, significant differences were observed with regard to the secondary products during fermentation according to the normal method and according to the method according to the invention with similar ethanol contents.

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Composition Fermentation with fermentation using free turning of enclosed cells · enclosed cells

Ethanol (mmol / L) 472 795 388 776

Glycerin (g / L) 3.87 4.67 4.18 5.48

higher alcohols (mg / 1) 117 200 70 107

Propanol (mg / L) 6.25 8.15 11.3 14.1

Isobutanol (mg / L) 38.8 63 7.8 19

Isoamyl alcohols (mg / l) 65.5 122 17.8 40.5

From this it can be seen that the rate of ethanoI formation can be multiplied by a factor of 5.

Example 6 Alcoholic fermentation of grape juice

In this example, the yeast fungus Schizosaccharomyces pombe, which has been cultivated under the same conditions as the yeast fungus Saccharomaces cerevisiae of Example 5, is used. One gets 1 1 of the nutrient medium for the preparation of 70 ml of polymer particles according to the method of Example 1. The advantage of this yeast is _s that it enables not only the fermentation of sugars to ethanol, but also the fermentation of malic acid to ethanol,

250 ml of white grape juice, which contains 126 g / l sugar and has a pH of 3.1, is fed to the reactor. This grape juice contains 12.5mMdl / l malic acid. The feed rate is 47 ml / hour, the ethanol concentration in the reactor is thus 600 »mol / ml and the effluent contains no more than 1.2 mmol / l malic acid. The level of productivity of this R _e is thus actuator 165 mmol / l · h χ

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Example 7 Alcoholic fermentation of sugar beet syrups (molasses)

In the case of the microorganism strain used in this example it is the technical yeast fungus Saccharomyces cerevisiae, which is resistant to fluoride, deposited at the Association Interprofessionnelle des Producteurs de Betteraves et d'Alcools de betteraves below the number COB 24-8.

This yeast is cultivated on a nutrient medium that consists of 2 1 Wickerham malt, the 5 ° g / l glucose and Contains 50 mg / 1 NaF. Taken after 4-8 hours of cultivation one the yeast obtained for the preparation of 70 ml Polymer particles. The reactor used is similar to that used in Example 2 and has a Volume of 250 ml. One introduces him to a solution of the following specified composition to:

Syrup (molasses) 286 g

Ammonium sulfate 2 g

NaF 40 mg

Water ad 1 1

pH 4.5, adjusted with phosphoric acid.

As a function of the amount charged, the following are obtained Productivity levels:

-T = 286 mmol / l. Hours. . D = 47 ml / hour

- T = 320 mmol / 1. Hour D * 65 ml / hour

In the examples described above, the Conversions do not require a great deal of ventilation, but the behavior of the acrylamide particles is satisfactory in the fermentation columns with blown air.

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The experiments described above were carried out in reactors because gases (CO ^) are released during these reactions v / ground, but if no gas formation occurs, they are preferably carried out in reaction columns.

The present invention permits the use of organisms entrapped in polymerizate particles Maintaining the activity of a purely enzymatic reagent. The inventive method is applicable to Execution of any enzymatic reactions, e.g. for the production of amino acids, for the synthesis of Antibiotics or for the synthesis of corticoids.

Example 8

Alcoholic fermentation of wine must, which is used to make beer

The microorganism strain used is the technical yeast Saccharomaces cerevisiae, deposited at the Ecole de Brasserie de Nancy under the number H 59.

This yeast is cultivated on a nutrient medium that 2 1 Wickerham malt with 10 g / l glucose f-at. After 48 hours From cultivation, the yeasts are removed to produce 70 ml of polymer particles, which contain the enclosed Contain yeasts.

The reactor used is identical to that used in Example 2 and is kept at ambient temperature. A wine must solution of 13.74 ° is introduced to him Plato with a pH of 4.95 too. The achieved Productivity levels are the following:

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- for a feed rate of 38 ml / hour: Φ = 167 mmol / l.h

- for a feed rate of 60 ml / hour: T = 218 mmol / l.hour.

The withdrawn solution contains about 80 mg of a higher alcohol per liter.

Example 9 Production of lactic acid

The material used for the implementation of the process micro-organism is the yeast Lactobacillus delbruckii ATCC 9649 · This microorganism is cultured at 4-O ⁰ C in the medium given below:

skimmed milk 100 g

Brewer's yeast extract 5 g

Glucose 100 g

Tomato juice 100 ml

Water to 1000 ml

pH value 7

4- 1 of the above medium are used to produce 70 ml of particles which contain the enclosed bacterium. The reactor used is identical to that used in Example 2 and it is kept thermostatically at a temperature of 4-5 ^° C. and with 2 η UaOH adjusted to pH 5.8. The following solution is fed to this reactor:

glucose after Skeegs and 1OO g / l Brewer's yeast extract 20 g / l Mineral salt solution 7th 09807 / 11 and Wright * 5 ml / 1 50

Sodium acetate 1 g / l

K ₃ HPO ₄ 2 g / l

With a charge of 180 ml / l, the degree of productivity is 21.5 g of lactic acid per liter.

Example 10 Denitrification by Hyphomicrobium

The microorganism strain used in this example is the Hyphomicrobium bacterium Hyphomicrobium W.O. from the Pasteur Institute. The bacterium is cultivated for 5 days in 4 1 milieu, the per 1 contains:

q..7H ₂ 0 2.13 g

_ ,. 2 ^s0 4 ° » ⁵ ε

MgS0 ₄ .7H ₂ 0 0.2 g

CaCl ₂ .2H ₂ O 10 mg

PeS0 ₄ .7H ₂ 0 5 mg

MnSO ₄ .4H ₂ O 2.5 mg

Na ₂ MoO ₄ .2H ₂ O 2.5 mg

KNO, 5 g

Methanol 0.5%

pH 6.7

The bacteria are then enclosed in a polymer matrix according to the procedure given in Example 1, thereby obtaining 30 ml of particles. The advantage of this bacterium is that it is denitrification using methanol as a carbon source permitted.

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The same medium as the nutrient medium is fed to the 250 ml reactor of Example 2, but it no _{longer contains (HEL) 2} S (V and its KtTO content has been brought to 3 g · per 1. This reactor is 2 months operated for a long time at a feed rate of 40 ml / hour. When converting the nitrates into nitrogen, a productivity of 20 mg nitrogen products per liter and per hour is obtained.

Example 11 Converting milk into yogurt

In the case of the microorganism strains Lctobacillus bulgaricus and Streptococcus thermophilus used in this example are strains that come from Laboratoires VISBT in lyophilized form and that For hours in 3 liters of the "Milk digested with papain" milieu with the composition given below (based on 1 l) can be regenerated:

Papain 1 g

Milk powder $ 0 g

Glucose 2 g

Tomato juice 100 ml

Tryptone 4 g

Brewer's yeast extract 6 g

NaH ₂ PO ₄ 6 g pH 7, adjusted with NaOH.

The bacteria are then placed in a polyacrylamide matrix (see Example 1) included, whereby 40 ml of particles are obtained.

In a 180 ml reactor, which is at a temperature of 40 ⁰ G

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is held, the mixed milk (milk powder + water) is introduced pH regulator is controlled, the limit value of which is pH 4.8 (so that the milk does not coagulate in the reactor). The reactor is operated for 10 days with an average feed rate of 120 ml / hour. and the acidity the milk is at 83 ° Dornic.

Example 12 Clarification through anaerobic digestion

The microorganisms used for this experiment are obtained from an anaerobic wastewater treatment plant in the city of Dijon The wastewater (sludge) is filtered and then centrifuged to concentrate the microorganisms. The procedure is as in the previous example, 170 ml of entrapped particles being placed in a 300 ml reactor which is kept at 35 ° C at a pH of 7 »2 will.

The milieu to be clarified has a DCO of I7 700 and the following specified composition:

soluble starch 8 g

Glucose 1 g

Brewer's yeast extract 3 β

Peptone 4- g

(HH ₄ ) ₂ S0 ₄ x 0.5 g

KH ₂ PO ₄ 0.57 S

1 g

: 7H ₂ O 100 mg

CaCl ₂ .2H ₂ O 7.5 mg

MnSO ₄ 10 mg

PeOl ₅ .6H ₂ O 0.5 ml

PH value ₇ 709807/1150

With a feed rate of 40 ml / hour. you get at the output a raw D.C.O. from 5800, the at In this process, sugars used an anthrone content from 9 »4- s / l to 0.47 g / l. By gas chromatography the formation of methane is detected.

Although the invention has been explained in more detail above with reference to preferred embodiments, it is It goes without saying for the person skilled in the art that it is by no means restricted to it, but that this is done in many ways Aspects can be changed and modified without thereby departing from the scope of the present invention is left.

Patent claims:

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Claims (1)

Claims Process for carrying out enzymatic reactions, in which microorganisms enclosed in a polymer matrix are used, characterized in that that one in a reactor which contains the microorganisms enclosed in a polymer matrix, continuously Introduces a culture medium for the microorganisms that contains the product (s) to be treated, and that the reaction products are continuously withdrawn, ensuring that the trapped microorganisms remain in the reactor. 2. The method according to claim 1, characterized in that the polymer matrix is a polyacrylamide matrix used. > 3 · Method according to claim 1 and / or 2, characterized in that that a bacterium or a yeast is used as the enclosed microorganism. 4-, method according to claim 3 »characterized in that bacteria of the genus Lactobacillus or Pseudomonas are used as the enclosed microorganism. 5. The method according to claim 3, characterized in that that the included microorganism is yeast of the genus Saccharomyces or Schizosaccharomyces used. 6. The method according to claim 3 and / or 4, characterized in, that Lactobacillus casei, Lactobacillus delbruckii or Pseudomonas aeruginosa is used. 709807/1150 7o method according to claim 5, characterized in that that one uses Saccharomyces cerevisiae or Schizosaccharomyces pombe as the enclosed microorganism. 8. The method according to at least one of claims 1 to 7 » characterized in that a mutant with low growth is used as the microorganism. 9v application of the method according to one of claims 1, 2, 3, 5 »7 and 8 for the production of ethyl alcohol from Solutions containing sugar 709807/1 150