DD236754A1 - PROCESS FOR THE PREPARATION OF GLUCOSE ACID BY BACTERIA - Google Patents

Abstract

The invention relates to a microbiological process for the production of free gluconic acid by oxidation of glucose. The goal is to develop a cost-effective and technologically simple process. The synthesis is catalyzed by using acidophilic methylotrophic bacteria. It is carried out under unsterile conditions in a p H range between 6 to 1, neutralization is not required. And since the enzyme is constitutive, the oxidation starts immediately after the addition of glucose, which allows for higher space-time yields than for A. niger. The productivity is up to 15 gh 1l 1 and it reaches concentrations up to 310 gl 1 and yields up to 97%. These values are also possible with biomass concentrations 5 gl 1. At these low biomass concentrations - in the case of the production of gluconic acid for technical purposes - complicated separation operations are eliminated.

Description

Fields 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 catalase) or electrochemically by oxidation of glucose (see HJ Rehm: Industrielle Mikrobiologie, 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, cluconobacter and pseudomonas species can also convert glucose into gluconic acid, bacteria are not used in industrial processes (see K.Yamada: Biotechn., Bioeng., 19 [1977] 1563; HJ Rehm: Industrial Microbiology, Springer-Verlag, Berlin-Heidelberg-New York, 1980). The known microwave methods are characterized in 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 pH value must be kept constant by titration with Al kali - or alkaline earth hydroxide between 6 and 7 (see Patent DE-AS 1817907) .This need for neutralization causes a further disadvantage insofar as by the resulting increase in viscosity considerable difficulties the further technical process (increased energy consumption, work-up, etc.) arise (see Patent DE-AS 1817907).

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 task of the invention is solved so that acidophilic methylotrophic bacteria species, eg. B. MB 58 IMET B 346, on methanol (or Glukr se) are cultivated according to the methods known for SCP synthesis, which bacteria synthesize a glucose-oxidizing enzyme. This bacterial biomass can be used as a protein source for feed or food purposes and / or as a producer of gluconic acid from glucose. The formation of gluconic acid can be carried out 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 leaching of the growth medium (eg methanol, NH ₃ ) and addition of glucose be carried out in the fermentation medium (glucose concentration 5 to 20%). The latter requires an introduced by the nutrient solution contamination with salts. Since the biomass (producer) is produced according to the chemostat principle under C-limitation, impurities due to intermediates of the metabolism are negligibly small. In addition, the biomass concentration in the production phase in the interest of high spec. Product formation rates (up to 6 g · h ^-1 · g ^-1 ) and yields (up to 97%) can be kept below 5 g · Γ ^1. This makes it possible, in the case of obtaining gluconic acid, for technical purposes, for example also in the washing industry, The simplest work-up, prior separation of the biomass is not necessary The aqueous solution of gluconic acid (up to 310g I " ¹ ) can be concentrated immediately by known methods. The advantage of using these bacteria is that they can produce non-sterile as a producer of gluconic acid, because growing in the acidic pH range, on methanol as a carbon and energy source, that the product formation immediately, ie without delay and not with as for Aspergillus niger necessary induction phase for the glucose-oxidizing enzyme (see patent

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DE-AS 1817907, US Pat. No. 3,669,840) in which the pH is optimal for growth, but also above pH4 to pH 6, after addition of glucose, which allows higher space / time yields compared to the known technical solutions ( up to 15g · h " ¹ · Γ ¹ ) The fact that the pH range of glucose oxidation far exceeds that for the growth of these acidophilic bacteria has 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 and continuously.

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 MB58 IMET B346 (see Example 5) used for the production of gluconic acid was separated from the culture fluid at 7000xg with a T-23 centrifuge, washed once with water and recentrifuged. The sediment was suspended in water. The pH of the suspension was 6.0.

100 ml of the suspension (1.4 g / l dry matter) and 20 g of glucose were placed in a 500 ml shake flask 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 was 5.2 g × ¹ h ^-1 .

Example 2

To a suspension of MB 58IMET B346 bacteria in water (50 g / l bacterial dry mass, pH 4.2) in a stirred tank reactor of 151 total volume at the beginning of 800g glucose monohydrate (starting concentration of glucose 15.4%), after 21 and 35 Hourly 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 × g " ¹ h" ¹ , the productivity was 14.6 g, and the yield was 92%. After 52 hours, 310 g / l gluconic acid was determined. The residual glucose in the fermentation medium was <0.1%. This corresponds to an average productivity of 6.0 g × ¹ h ^-1 , 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 MB58IMETB346 bacteria in water (1 g / l bacterial solids, pH 4.5) was added 2.4 kg glucose monohydrate in a stirred tank reactor of 30 μl total volume, 1.2 kg after 21 hours and 0.6 kg glucose monohydrate after 31 hours , The experiment was carried out analogously as described in Example 2.

The following values were determined: 121.5 g / l Gluconic acid formed after 21 hours: 95.4% Yield: 5.7 g-r 'h Productivity: 5,7g-g- ^1- l specific product formation rate: 4.4% Residual glucose: 210g / l Gluconic acid formed after 44.5 hours: 91.1% Yield: 5% Residual glucose: 4JG T ^ h average productivity: 4Jg ^ g- ¹ -! average specific product formation rate

Example 4

To an aqueous glucose solution analogous to Example 2, 1 g / l MB58 IMET B346 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.

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Example 5

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

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 -7H ₂ O 4,2mg / 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 6,0mg / l MnSO ₄ -4 H ₂ / 4.7 mg / 1 Yeast extract 0.1%

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

After setting a pH of 4.1 and a temperature of 32 ^C , the fermentation medium is inoculated with the culture MB 58 IMET B346, so that a starting concentration of about 0.5 g biomass / l results. The medium is gassed at a ventilation rate of 1001 / h and stirred at 1 200 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 O 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 bacterial mass MB58 IMET B346.

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. Stirrer speed and aeration rate during the production process is 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 approx.

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 / lh. 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 MPa at the start of 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.

In addition, the retention of the biomass or enzyme concentration required for the glucose oxidation process can be effected by carrier fixation or immobilization by one of the known processes.

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.

Claims (2)

ι. -1-463 34 Claim of invention:; 1. A process for the production of gluconic acid by means of bacteria, characterized in that glucose by acidophilic methylotrophic bacteria suitable for methanol based SCP synthesis, such as MB 58 IMET B346, under unsterile conditions, both for the production of the producer as well as in the product formation, and without neutralization during product formation in a pH range between 6 and 1 and at temperatures between 10 to 35 ° C to free gluconic acid is oxidized, the product formation even with low cell concentrations (to o, 5 g 1 ~ ¹ ), therefore, in the case of the production of gluconic acid for technical purposes separation of the biomass is unnecessary, and the product formation process can be closely coupled with a method for SCP synthesis. 2. The method according to item 1, 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.