RU2103346C1 - Fungus strain aspergillus niger - a producer of citric acid - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: new strain of fungus Aspergillus niger VKPM F-722 - a producer of citric acid. Strain is able to synthesize citric acid at 80% yield of ethanol content in medium and at 110% yield of a mixture of ethanol and sucrose. The use of this strain ensures to use sugar-containing media at increased ethanol level as carbon-containing substrate. EFFECT: new strain indicated above, increased yield of product.

Description

 The present invention relates to the field of microbiological industry and is a new non-pathogenic strain of the fungus Aspergillus niger (VSB-945 (2)), capable of synthesizing citric acid during deep cultivation.

 Microbiological citric acid is used in both food and light industries.

 Citric acid can be formed as a result of the metabolism of fungal, yeast, bacterial cultures (Journal of science, 1986, 51, n 2, Biotechnology, 1992, 1, pp. 46-49, German application N 3417777, UK application N 1603887).

 Food carbon sources are most often used as a carbon source in the biosynthesis of citric acid: sugar, molasses, and Aspergillus niger mushroom as producers. However, in terms of economic efficiency, the use of non-food carbon sources such as ethyl alcohol is preferable. Existing strains of microorganisms are not able to efficiently synthesize citric acid using industrial alcohol in high concentrations in the medium.

 The closest in biosynthetic activity is the strain Aspergillus niger F-710. When growing this culture on sucrose, the yield of citric acid is 90-95% (Avchieva PB, Vinarov A.Yu., Kozlova IA Biosynthesis of citric acid from glucose during continuous fermentation of Asporgillus niger. Applied biochemistry and microbiology. T. 29, 1993, issue 4, pp. 567-575), however, ethanol is synthesized by ethanol in ethanol with a yield of 50%, while the concentration in the medium of ethanol in excess of 2.5% inhibits the growth of the culture, which complicates the fermentation process .

 In connection with the stated objective of the present invention was the selection of an active non-pathogenic strain of the fungus Aspergillus niger, characterized by production properties, the ability to synthesize citric acid on ethyl alcohol and sucrose, high productivity, manufacturability during cultivation.

 The proposed new strain of the fungus Aspergillus niger F-722 was obtained by auto-selection of the strain Aspergillus niger F-710 on a mineral nutrient medium containing ethyl alcohol 4-6%, sucrose 3%, ammonium sulfate 2 g / l, potassium phosphate monosubstituted 1.5 g / l , magnesium sulfate 0.5 g / l, zinc sulfate, seven-water 0.01 g / l.

Cells were seeded with a two-day culture of Aspergillus niger F-710 fungus, 1 loop per liquid nutrient medium (100 ml). Cultivation was carried out with stirring, aeration, at a temperature of 30 ^o C for 45 days.

 During the cultivation process and at the end of cultivation, the content of citric acid was determined by the method of oxidative bromination based on the formation of pentabromoacetone followed by spectral analysis.

 The yves of the two suspensions obtained (4% ethyl alcohol and 6% ethyl alcohol) were seeded with wort-agar cultures and a quality medium supplemented with an indicator of bromocresol-green.

 Dilutions were selected so that 50-80 colonies grew. From each exposure, colonies were selected that were stained bright yellow. Selected 20 fungal cultures were grown on a nutrient medium of the above composition for 7 days. Citric acid content was determined in the culture fluid. Based on the results, a strain of Aspergillus nuger VSB-945 fungus was selected (2).

 Adapted to the consumption of ethanol in high concentrations, the strain was tested for the stability of the newly acquired properties. It was transferred to a liquid nutrient medium containing sources of mineral salts, nitrogen, phosphorus, as well as carbon sources: ethyl alcohol, sucrose, taken in various concentrations.

 When Aspergillus niger cells VSB-945 (2) were transplanted onto agar medium with a concentration of 4% ethyl alcohol, cells of the adapted strain form larger colonies than Aspergillus niger F-710 fungus cells after three days of growth.

 Determination of the systematic position of the selected strain, carried out in accordance with the determinant of fungi (Bergey s Manual of Systematic Bacteriology - Baltimore. Hong-Kong: London: Sydney, 1989, V. 1,2,3,4).

 Determining the degree of pathogenicity of the selected strain of Aspergillus niger confirmed the absence of pathogenicity.

 Characterization of the strain of fungi Aspergillus niger F-722.

Cultural traits: the wort agar colonies are white, furry, after three days brown spores are formed at T-30 ^o C. When grown on Chapek’s medium at T = 28 ^o C, pH 6.0 a branched fungal mycelium is formed without granular inclusions . There is no growth on MPA.

 Morphological signs: the mycelium consists of hyphae intertwined, colorless, the diameter of hyphae is 3-6 microns. With superficial growth, the fruiting organs rise - the conidiophores, which are thickened unseptic hyphae up to 2000 microns in length.

Physiological and biochemical signs: assimilates ethyl alcohol, sucrose, glucose. It does not need vitamins and growth factors. Calculation at a temperature of 20-40 ^o C, optimal values 28-30 ^o C, grows at pH 1.0-7.0, optimal values 5.8-6.2.

 The strain Aspergillus niger VSB-945 (2) was deposited in the All-Russian collection of industrial microorganisms of the State Genetics Research Institute under the number F-722.

The strain Aspergillus niger F-722 was tested in laboratory conditions in a fermenter Y 20 l, T = 130 ^o C, n = 400 rpm with aeration of 0.3 m ³ / h, PH 1.5-6.5 on a synthetic nutrient medium of the following composition: ethyl alcohol 3%, ammonium sulfate 2 g / l, monosubstituted potassium phosphate 1.5 g / l, magnesium sulfate 0.5 g / l, zinc sulfate, seven-water 0.012 g / l, for 30 hours, at Ethyl alcohol was added fractionally: the concentration of ethyl alcohol in the medium did not exceed 2.5%. Inoculum biomass was prepared by growing a two-day culture of mushrooms on the above medium.

 For a comparative evaluation, studies were carried out with the following strains of the fungus: Aspergillus niger F-710, Aspergillus niger F-228.

 When growing Aspergillus niger F-722 fungus on a nutrient medium containing ethyl alcohol, it synthesized citric acid with a yield of 80% of the supplied substrate, while the F-228, F-710 fungi strains showed low synthetic activity (the yield was 48% and 52% respectively).

 Information confirming the possibility of carrying out the invention.

Example 1. The strain of the fungus Aspergillus niger F-722 was cultured in a laboratory fermenter with a working volume of 20 l on a mineral medium, the following composition: ammonium sulfate 2 g / l, potassium phosphate monosubstituted 1.5 g / l, magnesium sulfate 0.5 g / l, zinc sulfate 0.012 g / l. 2% ethyl alcohol and 7% sucrose were added to the medium. Cultivation was carried out at T = 32 ^o C, ref. pH 6.5 with stirring 400 rpm, aeration of 0.4 m ³ / h, PH was not maintained. As a result of the experiment, 100 g / l of citric acid was synthesized, the yield was 110%.

 Example 2. The strain of the fungus Aspergillus niger F-722 was grown as in example 1, only in the nutrient medium contained 4% ethyl alcohol, 6% sucrose. The yield of the biosynthesis product, citric acid, was 93%.

 Example 3. The strain Aspergillus niger F-710 was grown as in example 1, while the yield of citric acid from the supplied carbon-containing substrates was 90%.

Thus, on the basis of the studies it can be concluded that the proposed strain of Aspergillus niger F-722 is an ethanol-assimilating producer of citric acid, characterized by the following distinctive features from the prototype Aspergillus niger F-710:
synthesizes citric acid in a nutrient medium containing elevated concentrations of ethyl alcohol and sucrose, while the yield from substrates is 90-110%;
stable in its morphological, cultural and physiological properties.

 Based on the data from the above studies, the strain of the fungus Aspergillus niger F-722 is recommended as a new producer of citric acid.

Claims (1)

 The strain of the fungus Aspergillus niger VKPM F-722 producer of citric acid.