DD236754B1 - PROCESS FOR THE PREPARATION OF GLUCOSE ACID BY BACTERIA - Google Patents

Description

Field of application of the invention

The invention relates to a biotechnical process for the preparation of an aqueous solution of gluconic acid by oxidation of glucose with the aid of bacteria.

Characteristic of the known technical solutions

Gluconic acid can be produced on an industrial scale microbially using molds or bacteria, biochemically with the aid of immobilized glucose oxidase (and catalytase) or electrochemically by oxidation of glucose (see HJ Rehm: Industrial Microbiology, Springer Verlag, Berlin-Heidelberg-New York 1967).

The commercial production of gluconic acid, which is currently used mainly in the food and pharmaceutical industries, is almost exclusively microbial, mostly strains of Aspergillus niger being used. Although the ability to oxidize glucose to form gluconic acid (and H ₂ O ₂ ) is also known in penicillia and pullularia and various aceto, gluconbacter and pseudomonas species can also convert glucose to gluconic acid, bacteria are not used in engineering procedures (see K.Yamada: Biotechn., Bioeng., 19 [1977] 1563; HJ Rehm: Industrial Microbiology, Springer Verlag, Berlin-Heidelberg-New York 1980). The known microbial processes are characterized by the fact that high gluconic acid concentrations and high yields are achieved. They all have the disadvantage that the production culture under sterile conditions - and only for the purpose of glucose oxidation - are obtained and that both in the growth phase and in the first product formation phase ("fermentation phase") - until the enzyme synthesis is completed - sterility and preserved the pH value must be kept constant by titration with alkali metal hydroxide or alkaline earth metal hydroxide between 6 and 7 (see Patent DE-AS 1817-907) .This need for neutralization causes a further disadvantage insofar as the increase in viscosity which results therefrom poses considerable difficulties for the further technical process (increased energy consumption, work-up, etc.) arise (see patent DE AS1817907).

Object of the invention

The object of the invention is to develop a process for the production of gluconic acid with little technological effort.

Essence of the invention

The invention has for its object to produce gluconic acid under unsterile conditions, without neutralization of the medium during the production of the producer and the product formation and without induction phase for the formation of the glucose-oxidizing enzyme.

According to the invention, the object is achieved by culturing acidophilic methylotrophic bacterial species by known methods on methanol or glucose and used as producers of gluconic acid by oxidation of glucose. Suitable microorganisms according to the invention are all acidophilic methylotrophic bacterial species.

Advantageously, the acidophilic methylotrophic strain Acetobacter methanolicus IMET B 346 can be used.

Other strains to be exemplified are Thiobacillus acidophilus DSM 700 or Thiobacillus versutus CCM 2505.

The microbial glucose oxidation takes place in the pH range of 6-1 and at temperatures between 10 and 35 ⁰ C, whereby free gluconic acid is obtained.

The formation of gluconic acid can be carried out either after separation of the biomass produced from the fermentation medium in an aqueous glucose solution, which has the advantage of high purity of the product and low workup operations, or directly after exhaustion of the growth medium (eg methanol, NH ₃ ) and addition of glucose be carried out in the fermentation medium, wherein the glucose concentration may be 5-20%.

Since the producer is produced according to the chemostat principle under C-limitation, impurities of gluconic acid by intermediates of the metabolism are negligibly small.

The biomass concentration in the production phase may be in the interest of high specific product formation rates (up to 6 g · h ^-1 · g ^"1) be maintained (to 97%) below 5 g · Γ ^1, to 0.5 g · Γ ¹ and yields. This allows - in the case of the production of gluconic acid for technical purposes, eg in the detergent industry -simple processing, a prior separation of the biomass is not necessary.

The aqueous solution of gluconic acid (up to 310g · Γ ¹ ) can be further concentrated by known methods.

The advantage of using acidophilermethylotrophic bacterial species is that they can be produced as non-sterile producers of gluconic acid on methanol as carbon and energy source, that the product formation immediately, ie without delay and not with the necessary as for Aspergillus niger induction phase for the glucose-oxidizing Enzyme (DE-AS 1817907, US 3669840) at the optimal pH for growth, but also in the range of pH 4-6 after addition of glucose, which allows higher space-time yields compared to the known technical solutions (up to 15 g · h ^-1 · Γ ¹ ) The fact that the pH range of the glucose oxidation far exceeds that for the growth of these acidophilic bacteria, gives further advantages.

The product formation can be carried out non-sterile and neutralization is not required, and thus the disadvantageous increase in viscosity in the known microbiological processes which produce gluconic acid substantially as gluconate can be avoided.

Finally, the production of gluconic acid for technical purposes requires no complex operations for separation because of the low biomass concentration required for production. The production of gluconic acid can be carried out both batchwise.

In the case of existing SCP production based on these bacteria, close coupling with gluconic acid production is advantageous in that the gluconic acid producer is immediately available.

embodiments example 1

The bacterial biomass MB 58 IMET B 346 (see Example 5) used for the production of gluconic acid was separated from the culture fluid at 7000 × g using a Τ-23 centrifuge, washed once with water and centrifuged again. The sediment was suspended in water. The pH of the suspension was 6.0.

100ml of the suspension (1.4g / l dry weight) and 20g glucose were placed in a 500ml chute! ко I be η given and shaken at 30 ° C for 40 hours under unsterile conditions. The gluconic acid concentration determined by titration or spectrophotometrically according to HU Bergmeyer (Methods of Enzymatic Analysis, Berlin, Akademie-Verlag, 1970) was 206.8 g / l; this corresponds to a yield of 97.5%. The residual glucose was 2.6%, the specific product formation rate was 3.7 g · g ^"1 · h" ¹ and the productivity of 5.2 g · Γ ¹ · h ^'1.

Example 2

To 4 l of a suspension of MB 58 IMET B 346 bacteria in water (50 g / l bacterial dry mass, pH 4.2) 800 g of glucose monohydrate (starting concentration of glucose 15.4%) after 21 and 35 hours further 500g glucose monohydrate added.

The following process parameters were adhered to:

Stirrer speed: 800 rpm Ventilation rate: 10 l / h

Temperature: 30 ^° C

A regulation of the pH value does not take place.

After 11 hours, 161 g / l gluconic acid were determined by spectrophotometric measurement (see Example 1). At this time, the specific product formation rate was 2.9 g- ^1, the productivity was 14.6 g and the yield was 92%, and 310 g / l gluconic acid was determined after 52 hours, and the residual glucose in the fermentation medium was <0.1% corresponds to an average productivity of 6.0 g Γ ¹ · h · an average specific product formation rate of 1.15 g · g · ¹ · h ^-1 and a yield of 89.9%. The oxygen requirement for the oxidation of the glucose was 0.12 g oxygen / g gluconic acid in the first 20 hours and increased to 0.16 g oxygen / g gluconic acid in the following 30 hours.

Example 3

To a suspension of MB 58 IMET B 346 bacteria in water (1 g / l bacterial dry mass, pH 4.5) was added to a stirred tank reactor of 3 liters total volume 2.4 kg glucose monohydrate, after 21 hours 1.2 kg and after 31 Hours 0.6kg glucose monohydrate added. The experiment was carried out analogously as described in Example 2.

The following values were determined:

Gluconic acid formed after 21 hours: 121.5 g / l Yield: 95.4%

Productivity: 5.7-l " ¹ -h" ¹

specific product formation rate: 5.7 g · g " ¹ · h" ¹

Residual glucose: 44%

gluconic acid formed after 44.5 hours: 210 g / l

Yield: 91.1%

Residual glucose: 5%

average productivity: 4.7 · Γ ¹ · h " ¹

average specific

Product formation rate: 4,7g-g " ¹ 'h" ¹

Example 4

To an aqueous glucose solution analogous to Example 2, 1 g / l MB 58 IMET B 346 bacterial biomass is added and fermented at the same process parameters. After reaching a gluconic acid concentration of about 100 g / l again 1 g biomass / l is added. The addition of another 1 g of biomass / l takes place after reaching an acid concentration of 250 g / l. After a total fermentation period of 60 hours, the product formation process is terminated at an acid concentration of 359 g / L corresponding to a space-time yield of 5.98 g / Lh. The substrate yield is 89.8% and the average oxygen requirement is 0.12 g / g acid.

Example 5

In a laboratory stirred tank fermentor of 121 gross volumes with 6 leaf disc stirrers are added 51 of a nutrient medium following the composition:

NH ₄ Cl 2.0 g / l H ₃ BO ₃ 7.0 mg / l K ₂ HPO ₄ 0.6 g / l FeCl ₂ 7 H ₂ O 4.2 mg / l MgSO ₄ .7H ₂ O 0.4 g / l CaCl ₂ -6H ₂ O 7,2mg / l CuSO ₄ -5H ₂ O 2.4 mg / 1 Na ₂ MoO ₄ -2H ₂ O 7.8 mg / l CoSO ₄ -5H ₂ O 0.6 mg / l ZnCl ₂ -7H ₂ O 6.0 mg / l MnSO ₄ -4H ₂ O 4.7 mg / 1 Yeast extract 0.1%

This nutrient medium allows an organism increase of 5g / l.

After setting a pH of 4.1 and a temperature of 32 ⁰ C, the fermentation medium is inoculated with the culture MB 58 IMET B 346, so that there is a starting concentration of about 0.5 g biomass / l. The medium is gassed at a ventilation rate of 1001 / h and stirred at 1200 rpm. By addition of 15 g of methanol / l of the growth process is started and the methanol concentration is maintained by adding portions in the range between 0.2 and 0.4%. The regulation of the pH is carried out by means of 5% sodium hydroxide solution. After reaching a biomass concentration of about 5.5 g / l, limited by the nitrogen content of the fermentation medium, the discontinuous growth process is terminated and added to the biomass-containing nitrogen-free fermentation medium 200g / l glucose. At the same time, the stirrer speed is reduced to 800 rpm and the aeration rate to 40 l / h.

After a product formation time of 20 and 30 hours, in each case a further 125 g of glucose / l are added to the fermentation medium. The final gluconic acid concentration reached is 308 g / l after 44 hours, corresponding to a yield of 89% and a space-time yield of 7.0 g acid / lh

The average oxygen demand required for glucose oxidation is 0.13 g 0 ₂ / g acid.

Example 6

121 ml of glucose aqueous solution (100 g / l) are introduced into a 3 l stirred-tank reactor in the reactor and inoculated with 7 g / l of MB58 IMET B346 bacterial mass.

Analogously to Example 3, 1.2 kg or 0.6 kg of glucose are added to the fermentation medium after 21 hours and 31 hours. The stirrer rotation rate and the aeration rate during the production-forming process are 100 rpm and 1.41 rpm, respectively. After a fermentation time of 45 hours, a gluconic acid concentration of 215 g / l is reached. At this time, the continuous metered addition of a 25.0% aqueous glucose solution with a residence time of 20 hours. After about 1.5 residence time periods, a steady-state process state occurs with an average gluconic acid concentration of 227 g / l, corresponding to a productivity of 11.3 g acid / l h. The gluconic acid-containing aqueous fermentation medium is removed cell-free from the reactor via a suitable membrane filter continuously. For this purpose, the reactor pressure is increased to 0.5 M Pa at the beginning of the continuous operation. The glucose residual concentration in the cell-free aqueous gluconic acid solution is 0.5%.

After a continuous process of about 300 hours, the process is interrupted and restarted with fresh biomass.

The retention of the biomass or enzyme concentration required for the glutose oxidation process can also be achieved by carrier immobilization or immobilization according to one of the known methods.

A periodic renewal of the biomass can be avoided if the required for product formation biomass is continuously fed to the process analogous to a two-step process. In this case, however, a downstream work-up process is required to obtain a cell-free aqueous gluconic acid solution.

Example 7

To a 15% glucose solution 25 g / l of the bacterial culture Thiobacillus versutus CCM 2505, cultured on methanol as the sole source of carbon and energy, and fermented analogously to Example 2.

After 35 hours, a gluconic acid concentration of 142 g / l, corresponding to a productivity of 4.05 g acid / l h and a yield based on glucose of 94.0%. The residual glucose concentration is 0.85 g / l.

A regulation of the pH value did not take place. At the end of the process, the pH of the medium was 1.9.

Example 8

100 ml of a suspension containing 10 g of glucose and 5 g biomass of the bacterial culture Thiobacillus acidophilus DSM 700 is placed in a 500 ml shake flask and cultured at 32 ⁰ C on a shaker. The bacterial culture was previously cultivated non-sterile on methanol as a substrate and separated from the fermentation medium by centrifugation. The pH of the suspension in the shake flask was 6.1 at the beginning of the cultivation. After a cultivation time of 50 hours, a free gluconic acid concentration of 91 g / l was determined titrimetrically. This corresponds to a productivity of 1.8 g / lh and a yield of 91%. The residual glucose concentration was 1 g / l. The pH at the end of the non-sterile cultivation phase was 2.3.

Claims (5)

claims: 1. A process for the production of gluconic acid by means of bacteria, characterized in that glucose by means of acidophilic methylotrophic bacteria under unsterile conditions, both for the production of the producer and in the product formation, and without neutralization during product formation in a pH range between 6 and 1 and at temperatures between 10 and 35 ⁰ C to free gluconic acid is oxidized, wherein the product formation with cell concentrations of 0.5 g 1 ~ ^{1 is} performed. 2. The method according to claim 1, characterized by the use of the bacterial strain Acetobacter methanolicus IMET B 346th 3. The method according to claim 1, characterized by the use of the bacterial strain Thiobacillus acidophilus DSM 700th 4. The method according to claim 1, characterized by the use of the bacterial strain Thiobacillus versutus CCM 2505. 5. The method of claim 1 and any one of claims 2-4, characterized in that the product formation takes place either by adding glucose to the growth medium containing the producers, or by adding the producer to an aqueous glucose solution.