DD259969A3 - PROCESS FOR THE PREPARATION OF GLUCONIC ACID BY BACTERIA - Google Patents

Abstract

The invention increases the oxidation performance of the bacterial biomass used as a catalyst for microbial glucose oxidation. This is done by phenotypic measures taken during or at the end of the growth phase of the bacteria. For this purpose, the bacteria are shocked with methanol or glucose or the culture medium, if it is not glucose, but z. As methanol or acetate, gluconic acid, glucanate or glucose is added and / or held constant the pH value in the production phase by titration. Through these measures, both significantly higher productivity and higher acid or Gluconatendkonzentrationen be achieved. This significant increase in process efficiency is also achieved using low cell concentrations.

Description

Field of application of the invention

The invention relates to a biotechnological process for the preparation of an aqueous gluconic acid or gluconate solution by oxidation of glucose by means of bacteria.

Characteristic of the known technical solutions

The industrial production of gluconates or gluconates, which are currently used in large quantities in the food and pharmaceutical industry and used in detergent production or as industrial cleaners due to their complex-forming properties, takes place almost exclusively by microbial route, mostly strains of Aspergillus niger (JH Rehm: Industrial Microbiology, Springer Verlag, Berlin-Heidelberg-New York 1980).

Although it is known that various Acetobacter, Gluconobacter, and Pseudomonas species are capable of oxidizing glucose to gluconic acid (PE Milsom, JL Meers: Gluconic and Itaconic Acids., In Comprehensive Biotechnology [Ed M. Moo-Young], Vol. 3 [ed. By CL Cooney, AE Humphrey], Pergamon Press, Oxford 1985, pp. 681-700), only species of the genus Gluconobacter are used in technically implemented methods.

The technical processes are carried out discontinuously, in two stages. All have the disadvantage that the producers can be obtained only under sterile conditions, even during the product formation phase of the sterility must be largely maintained and used as a substrate for the production of the producer glucose. The latter is required to raise product production performance to a high level.

DD-WP 238067 discloses a continuous process using bacteria from Acinetobacter calcoaceticus, which avoids this by using a substrate mixture. The constantly present glucose keeps the oxidizing potential of the producer high, without glucose being assimilated. It can maximaj be converted to gluconic acid. A disadvantage of this, as well as all neutrophil bacterial processes, is that the pH must be maintained between 5 and 7, preferably 7, by titration with alkali or alkaline earth hydroxide, and only at the end of the product formation phase in two-stage operation may decrease to lower values, as described in DE-AS 1817907. Although the method according to DD-WP 236754 does not have these disadvantages, the possible productivity of the overall process (with an achievable gluconic acid concentration of 310 g / l) does not exceed a value of 7 g acid / lh. The productivity of 15 g / lh in a concentration range up to 160 g / l described in this patent can only be achieved if a biomass concentration of 50 g / l is used. With such high biomass concentrations, however, several disadvantages are associated. So z. As the glucose-related yield at high cell concentrations by inhalation / endoxidation of glucose below 90%. There is additional effort for the production of biomass as a producer and for the separation of the biomass from the gluconic acid fermentation solution. The associated costs significantly burden the process.

Object of the invention

The object of the invention is to increase the oxidation performance of gluconic acid producers and thus to improve the productivity of processes for microwave gluconic acid production.

Essence of the invention

The invention has for its object to increase the capacity of bacteria for the oxidation of glucose to gluconic acid. According to the invention, the object is achieved in that the oxidation performance of the bacterial cultures used is increased by phenotypic measures at the end or during the growth phase.

In this case, it is advantageously carried out in such a way that the bacteria capable of producing gluconic acid of the genera Aceto- or Gluconobacter and Acinetobacter are exposed to methanol in concentrations of 1 to 10 g during or at the end of the carbon-limited cultivation for producing the producer over a period of 1 to 15 hours. l or are shocked with glucose up to 80 g / l, or that these strains, if they can not be cultured on glucose or, when growing on other carbon sources, (such as methanol or acetate), gluconic acid, glucanate or Glucose is added in a concentration between 0.01 to 0.5%. -

This procedure is distinguished from the prior art represented by DD-WP 236754 by an increased specific product-forming capacity. In the case of Acetobacter methanolicus, productivities of 26 g / lh in the region of the product formation phase up to 100 g acid / l or 17 g / l · h in the range up to 200 g acid / l can be achieved. It should be emphasized that these high productivities are already possible with biomass concentrations in the range between 0.5 to 8 g / l and that up to 400 g gluconic acid per liter can be achieved with a "everywhere" productivity of 10 g / lh, which significantly improves the economics of the process becomes.

The increased productivity in this way (i.e., by shock or addition of gluconic acid or glucose into the growth medium) can be further increased by keeping the pH constant during the product formation phase. Taking advantage of the advantages of non-sterile process control in the use of acidophilic bacteria both in the stage of biomass production and the actual production, the production of gluconic acid is preferably carried out with Acetobacter methanolicus and the pH is kept constant between 2.5 and 3.3, so that Productivities up to 400g acid / l of 7 g / l · h can be achieved. When using nonacidophilic bacteria, the pH should not fall below 4.5. By combination of shock or glucose or gluconic acid addition at the end of the growth phase and titration, a further increase in process productivity is possible, ranging from 200 g acid / l to 20 g / l · h and up to 400 g acid / l to 10 g / lh amounts.

Acid end concentrations of 500 g / l can be produced in this way. By following examples, the invention will be explained in more detail.

embodiments Example 1:

Acetobacter methanolicus is cultured in a 101 laboratory fermenter under the following conditions:

Reactor content 4kg

Residence time: 7h

pH value: 4.0

gel. O ₂ concentration: §5% of the saturation value

Substrate: methanol, C -impeded

Temperature: 32 ° C

Stirrer speed: 1,500 rpm

Nutrient solution composition: (weighted for 20g BTS / I)

NH ₄ CI 8g / l, K ₂ HPO ₄ 2.4 g / l, MgSO ₄ .7H ₂ O 1.6 g / l, CuSO ₄ 5H ₂ O 9,6mg / l, CoSO ₄ · 5H ₂ O 2.4 mg / 1, MnSO ₄ 4H ₂ O 18.8 mg / l, H ₃ BO ₃ 28 mg / l, FeCl ₂ · 7H ₂ O 16.8 mg / l, CaCl ₂ · 6H ₂ O 28.8 mg / l, Na ₂ MoO ₄ · 2H ₂ O 31.2 mg / l, ZnCl ₂ · 7H ₂ O 24 mg / l

After reaching a stationary continuous process state at a biomass concentration of 19.5 g / l and carbon-limited process control, the methanol content in the fermentation medium is set to 4.5 g / l and kept constant over a period of 5 hours. ·

After 5 hours of methanol shock, the biomass is harvested, centrifuged and used as a seed for microbial gluconic acid production.

For this purpose, 41% of a 15% strength glucose solution are added to a 121 stirred tank reactor and inoculated with Acetobacter methanolicus (methanol shocked) to give a cell density of 6.5 g / l. The product synthesis is carried out at the following process parameters:

Stirrer speed: 800 rpm

Temperature: 32 ° C

Ventilation rate: 50 l / kg · h

A regulation of the pH value during the process does not take place.

The addition of glucose is such that a current concentration range of S10 S 30g / l is maintained. The following results are obtained in comparison to unstocked biomass:

reaction Säurekonzen- 85 * productivity 17 * specific 2.6 * Time   tration 130 * 10.8 * 1.7 * 300 * 7.5 * product education 1.2 * IHI / G / l / / G / l-h / rate 5 115 23 / G / g-h / 12 200 16.6 3.5 40 390 9.8 2.6 * Use of unshocked biomass 1.5

The addition of glucose is controlled in the last 10 hours of the process so that at the end of the reaction the glucose concentration in the medium is <0.1%.

Example 2:

Analogously to Example 1, the strain Acetobactermethanolicus MB 58 is cultivated continuously in a 121 stirred tank reactor. Instead of pure methanol as a substrate, a mixed substrate consisting of methanol and gluconate in a ratio of 5: 1 is used. The biomass thus produced is used as described in Example 1 for microbial glucose oxidation in a laboratory fermenter. The cell concentration was 7.9 g Γ ¹ . The following values were determined:

reaction acid Concentration productivity specific Time tration / G / l-h / Product formation rate IHI / G / l / 20.6 / G / g-h / 5 103 15.5 2.6 10 155 13.0 1.9 20 260 9.5 1.6 40 380 1.2

The glucose-related yield was 93.2%, the residual glucose concentration in the medium <0.1%.

Example 3:

Analogously to Example 1, the strain Acetobactermethanolicus MB 58 is continuously cultured on the substrate glucose in a 121 laboratory stirred tank reactor. Biomass is separated from the fermentation medium in a laboratory centrifuge and, as described in Example 1, 4I of a 15% glucose solution in a 121 laboratory fermenter are inoculated, the concentration in the medium is 0.9 g / l.

The following values were reached in the product formation phase:

Reaction acid specific

time concentration productivity Product formation rate

/ h / '/ g / l / / g / l - h / / g / g-h /

12 190 26 260 36 310

15.8

10.0

8.6

17.6

11.1

9.6

The glucose-related yield was 94.3% ", the residual glucose concentration in the medium <0.2%.

Example 4:

Analogously to Example 1, the culture Acetobacter methanolicus continuously produced carbon-limited to methanol, harvested and used after centrifugation as a producer for Gluconsäuresynthese.

In deviation from Example 1, the pH of the fermentation medium is kept constant at 3.0 by titration with 50% sodium hydroxide solution during the process of product formation (used biomass concentration 6.5 g / l).

The following results are obtained:

Reaction acid specific

time concentration * Productivity Productification Rate

lh / / g / l / g / lh / / g / gh /

5 112 12 180 40 350 50 385 60 420 80 440

22.4 15.0 8.8 7.7-7.0 5.5

3.4 2.3 1.3 1.2 1.1 0.8

* calculated on free acid

The glucose-related yield is 94.1%.

Example 5:

Biomass of the culture Acetobacter methanolicus is produced analogously to Example 1 and shocked with methanol. Subsequently, the thus treated biomass is analogous to Example 4, d. H. under titration at a pH of 3.0, used for product formation. Under these conditions, the following results are obtained:

reaction Säurekon productivity specific Time concentration * / g / l · h / Product formation rate IHI / G / l / 25.0 Vg / g-h / CJL 125 20.0 3.8 10 200 17.3 3.1 20 345 10.4 2.7 40 415 8.1 1.6 60 485 1.2

* calculated on free acid

The glucose-related yield is 93.8%.

Examples:

Acinetobacter calcoaceticus ZIMET 11089 is cultivated in a laboratory fermentor at 30 ^c C, the pH value of 6.8 is kept constant by addition of NaOH or HCl. The dissolved oxygen concentration was set to greater than 20% saturation. The continuous cultivation was carried out on Na acetate as carbon source and a mineral salt medium of the following composition (in mg / l):

Ammonium Chloride 3400

Potassium hydrogen phosphate 310

Magnesium sulfate χ 7H ₂ O 15

Calcium chloride χ 2 H ₂ O 20

Iron (II) sulphate x7H ₂ O 5

Zinc sulfate χ 7 H ₂ O 0.44

Manganese sulphate χ 4 H ₂ O 0.82

Copper sulfate x 5 H ₂ O 0.78

Sodium molybdatex2H ₂ O 0.25

After reaching a steady state continuous process state, glucose is added to the fermentation medium and the glucose concentration is kept constant in a range between 5 and 30 g / l over a period of 5 hours.

After 5 hours of glucose shock, the biomass was harvested and used as in Example 1 for microbial gluconic acid production. Gluconic acid concentrations of 265g / l are reached after 40 hours.

With unshocked biomass, only 200 g / l gluconic acid was obtained in comparison.

Example 7:

Analogously to Example 6, the culture Acinetobacter calcoaceticus ZIMET 11089 was carbon-limited to acetate and then shocked with gluconate.

Gluconic acid concentrations of 237 g / l were obtained with this biomass after 40 hours.

Claims (6)

1. A process for the production of gluconic acid by means of bacteria using strains of the genera Acetobacter, Gluconobacter or Acinetobacter, characterized in that the oxidation performance of the bacterial cultures is increased by phenotypic measures during or at the end of the growth phase. 2. The method according to claim 1, characterized in that the oxidation performance is increased by a 1 to 15-hour substrate shock at the end of the carbon substrate-limited growth to produce the producer. 3. The method according to claim 2, characterized in that methanol are added to the shock in a concentration of 1 to 10g / l or glucose in a concentration to 80g / l. 4. The method according to claim 1, characterized in that, if the producer is not produced on glucose, the culture medium (eg methanol or acetate) gluconic acid, gluconate or glucose in a concentration between 0.01 to 0.5g / l, preferably 0.1 g / l are added. 5. The method according to claim 1, characterized in that in the production phase, the pH is kept constant by titration using Acetobacter methanolicus between 2.5 and 3.3 and of Acinetobacter calcoaceticus between 5 and 6.5. 6. Process according to Claims 1 to 3, characterized in that the producer is shocked and / or produced with the addition of gluconic acid, gluconate or glucose and the pH during product formation when using Acetobacter methanolicus 2.5 does not fall below and between 2.5 and 3.3 is kept constant.