RU2183218C2 - Strain of fungus aspergillus niger vkpm f-790 as producer of gluconic and citric acids - Google Patents

Abstract

FIELD: biotechnology, microbiological industry. SUBSTANCE: invention relates to a novel strain of fungus Aspergillus niger VKPM F-790. On depending of conditions the strain produces citric acid or its salts and gluconic acid (as its salts or gluconolactones). The strain VKPM F-790 ensures to rearrange manufactures using fungus Aspergillus niger for biosynthesis from production of monoproduct to production of some products. The novel strain shows the enhanced stability in acid media. EFFECT: improved economy efficiency, simplified production of gluconic acid. 1 tbl

Description

 The invention relates to the microbiological industry and relates to a new strain of the Aspergillus niger VKPM F-790 fungus, which produces, depending on the conditions created, citric acid (its salts) or gluconic acid (its salts or gluconolactones).

 Known strains of the fungus Aspergillus niger VKPM F-501 and Aspergillus niger VKPM F-171. Both strains are intended exclusively for the biosynthesis of citric acid under industrial conditions on sugar containing media of various compositions [1, 2].

 During the biosynthesis of citric acid with these strains, about 10% of gluconic acid is formed to the sum of all biosynthesis acids, which is from 7 to 8% relative to the introduced sugar. For the target production of gluconic acid or its derivatives, such indicators of biosynthesis are of no interest.

 Providing special conditions favorable for the formation of gluconic acid or its derivatives [3], the yield of these products increases to 75–85% of the added sugar. As by-products, citric and oxalic acids are formed, which make it difficult to isolate the target product and are lost irrevocably.

 The objective of the present invention is to obtain a new strain of Aspergillus niger fungus with high activity and productivity both in the biosynthesis of citric acid (its salts) and in the process of producing gluconic acid (its salts or gluconolactones) under industrial conditions.

 The new strain was obtained by selection from a strain of the Aspergillus niger VKPM F-171 mutant (hereinafter referred to as strain L-4) [2].

 For five years, under the conditions of industrial production of citric acid, sampling, sieving, and isolation of samples of the L-4 culture were carried out, subjected to mutations under natural conditions of production, which were accompanied by increased acid formation, increased resistance to bacterial and yeast infection.

 Particular attention is attracted to culture samples that provide, along with high technical and economic indicators in the biosynthesis of citric acid, their significant increase compared with the original strain L-4 upon receipt of gluconic acid and its derivatives.

 The value of the target biosynthesis products, the production of which is possible based on the use of a new strain, allows us to judge the information presented in [4].

 The strain of Aspergillus niger obtained as a result of selection has a collection number VKPM F-790.

 Cultural and morphological characters are studied in five-day-old cultures grown on Chapek's synthetic medium or wort-agar nutrient medium.

 Cultural traits in the environment of апapek.

 The colony has a diameter of 78 ± 2 mm, the color is beige, in the center is yellowish, raised above the substrate. The conidial heads in the center of the colony are large, densely located, form an elevation, their size decreases to the edge. The size of the asporogenic zone is about 6 mm. The reverse side of the colony is folded, white.

 The table summarizes the performance of the newly obtained strain VKPM F-790 in comparison with the known strain L-4.

 As can be seen from the table, on the Chapek medium, the VKPM strain F-790 has a different color and appearance of the reverse side of the colony.

 Cultural traits on wort-agar culture medium.

 The colony has a diameter of 95 ± 1 mm. The color is beige. Aerial mycelium is highly developed. In the center, the conidial heads are larger, densely arranged, fins to the edge. The size of the asporogenic zone is 14.5 ± 1.5 mm. The reverse side of the colony is folded, white.

 In contrast to the known strain (see table), the VKPM F-790 strain characterizes a larger colony size, an increase in the asporogenic zone and folding of the reverse side of the colony.

 Morphological characters are identified in five-day-old crops grown on Chapek's environment.

 Conidial heads are round in shape, their diameter is 110 ± 61 μm, bloating conidiophores spherical shape with a diameter of 31 ± 4 μm. Sterigmas are two-layer: first-order sterigms 15 ± 5 μm long, second-order sterigms 7.5 ± 2.5 μm long, their surface is covered with outgrowths. Conidia are connected in chains by flattened sides devoid of outgrowths. The average conidia size is 3.3 ± 0.3 μm. The conidiophores are straight, colorless, the length of the conidiophores is 867 ± 561 μm, and the diameter is 10.5 ± 3.5 μm.

 Comparison with the known strain L-4 shows that the VKPM strain F-790 has large sizes of conidial heads, smaller swelling of conidiophores, a length of first-order sterigm, size of conidia and diameter of conidiophores (see table).

 Among the biochemical properties of the VKPM strain F-790, its ability to grow and biosynthesis in more acidic media than the original strain deserves mention. During deep cultivation, the newly obtained VKPM strain F-790 has a pH optimum for gluconic acid biosynthesis from 3.5 to 6.0, in contrast to the known strain L-4 (optimum pH for 5.5-6.0 similar application). Probably, it is this feature of the VKPM F-790 strain that provides it with greater resistance to bacterial and yeast infection compared with L-4.

 The new VKPM strain F-790, like the well-known L-4, is aerob, prototroph. Able to provide fermentation from 20 hours to 12 days. It is well stored and reproduces its properties in the form of dry conidia (residual humidity 5-10 rel.%).

 The activity and productivity of the new VKPM F-790 strain is evaluated under industrial (biosynthesis of citric acid and calcium gluconate) and laboratory (biosynthesis of citric acid and gluconolactone salts).

 By activity is meant the yield of the target product obtained in relation to the introduced sugar for a certain fermentation time. The productivity of the process is judged by the volume of the biosynthesis product per unit volume of reactor per day.

As can be seen from the table, at close values of the activity and productivity of the new and known strains in the biosynthesis of citric acid, VKPM strain F-790 is 17-22% more active and 25-30 kg / m ³ • more productive than L-4 in the production of gluconic acid or its derivatives. These indicators accompany lower concentrations of biosynthesis by-products (see table).

 Attitude to nitrogen sources.

 Absorbs nitrogen from mineral salts and organic compounds: proteins, peptides, amino acids.

 Relation to carbon sources.

 Absorbs sucrose, glucose, fructose, sorbose, maltose; poorly utilizes dextrin, starch, lactose, galactose.

 The invention is illustrated by some specific examples of the use of strain VKPM F-790.

 Example 1

 Getting citric acid.

 1.1. Obtaining seed in the form of conidia.

Conidia are grown in thermostat chambers with controlled temperature and humidity on cuvettes filled with a 1.5 cm layer of nutrient medium composition, g / dm ³ :
Beer Wort - 20
Urea - 1
Sodium Chloride - 20
Agar - 20
Artesian Water - Else
pH before sterilization 5.8.

The medium is inoculated with a culture of strain F-790 and incubated for 7 days under conditions of forced aeration with sterile air, supporting the following growing regimen:
1-4 days: humidity - decrease from 80 to 60 rel.%;
5-7 days: humidity - decrease from 60 to 40 rel.%.

On day 8, the air temperature is reduced to 18 ^o C and conidia are collected by a vacuum device. Conidia are dried at 32 ^{° C.} to a residual moisture content of 7 rel.%.

 1.2. Soaking conidia.

4 g of conidia is soaked in 1 DM ³ sterile aqueous molasses solution containing 50 g of molasses.

Maintain a suspension of conidia on a rocking chair with a number of swings of 160 per minute for 7 hours at 34 ^o C.

 1.3. Cultivation of seed - inoculum.

In an industrial seed fermenter - inoculator with a volume of 10 m ³ prepare a nutrient medium of the following composition, g / DM ³ :
Molasses - 50
Potassium Ferrocyanide - 0.25
Ammonium Oxalate - 2
Zinc Sulphate - 0.005
Magnesium Sulfate - 0.25
Potassium Phosphate Monosubstituted - 0.16
Artesian Water - Else
A swollen suspension of conidia is inoculated on the prepared nutrient medium.

Next, the seed, inoculum, is grown using the following air-temperature conditions:
time up to 12 hours: temperature 38 ^o C, air flow 200 m ³ / h;
time up to 24 hours: temperature 37 ^o C, air flow 300 m ³ / h.

 1.4. Basic fermentation.

In an industrial fermenter with a volume of 100 m ³ , 60 m ^{3 of the} nutrient medium of the following composition is prepared, g / dm ³ :
Molasses - 60
Potassium Ferrocyanide - 0.25
Ammonium Oxalate - 2
Zinc Sulphate - 0.005
Magnesium Sulfate - 0.25
Potassium Phosphate Monosubstituted - 0.16
Artesian Water - Else
pH 5.5.

The inoculum is transferred to the fermenter and biosynthesis is carried out for 5 days, maintaining the following air-temperature conditions:
Day 1: air consumption 1200 m ³ / h, temperature 37 ^o C;
day 2: air flow 1500 m ³ / h, temperature 36 ^o C;
Day 3: air consumption 1800 m ³ / h, temperature 36 ^o C;
Day 4: air consumption 2000 m ³ / h, temperature 34 ^o C;
Day 5: air consumption 2200 m ³ / h, temperature 34 ^o C.

Starting from 3 days, topping up is made of 0.25 m ^{3 of the} nutrient medium of the following composition, g / dm ³ :
Molasses - 500
Potassium Ferrocyanide - 0.5
Ammonium Oxalate - 3
Artesian Water - Else
After separation of the biomass, 68 m ^{3 of a} solution with a total acidity of 10 wt.% And a concentration of citric acid of 9 wt.% (6120 kg) is obtained. The yield to spent sugar (8100 kg) is 75.6%.

 Example 2

 Obtaining sodium citrate.

 2.1. Obtaining seed in the form of conidia, as in example 1.

 2.2. Soaking conidia.

0.1 g of conidia is soaked in 700 cm ^{3 of} molasses water containing 40 g of molasses.

The suspension is kept on a rocking chair with a number of swings of 160 per minute for 7 hours at 34 ^o C.

 2.3. Basic fermentation.

In a laboratory fermenter with a volume of 10 dm ³ , 7 dm ^{3 of a} nutrient medium of the following composition is prepared, g / dm ³ :
Sucrose - 150
Zinc Sulphate - 0.005
Magnesium Sulfate - 0.25
Diammonium phosphate - 0.4
Potassium Phosphate Monosubstituted - 0.16
Urea - 2
Artesian Water - Else
A swollen suspension of conidia is seeded on a sterile growth medium.

Next, they conduct the process using the following air-temperature conditions:
day 1: temperature 38 ^o C, air flow 100 dm ³ / h,
day 2: temperature 36 ^o C, air flow 150 dm ³ / h,
3-5 days: temperature 34 ^o C, air flow 220 dm ³ / h.

 A solution of 10 wt.% Sodium hydroxide, starting from 3 days, maintain a pH of about 3.5.

 At the end of sugar utilization, the process is stopped, the biomass is separated, the pH of the solution is adjusted to 6.5. 810 g of sodium citrate are obtained. The yield relative to the sugar used (1070 g) is 75.7%.

 Example 3

 Obtaining calcium gluconate.

 3.1. Obtaining seed in the form of conidia, as in example 1.

 3.2. Cultivation of seed - inoculum.

In an industrial seed fermenter - inoculator with a volume of 1 m ³ , 600 dm ^{3 of the} nutrient medium of the following composition is prepared, g / dm ³ :
Glucose - 25
Magnesium Sulfate - 0.2
Manganese Sulfate - 0.05
Diammonium phosphate - 0.4
Monosubstituted potassium phosphate - 0.2
Artesian Water - Else
A swollen conidia suspension is inoculated onto a prepared sterile growth medium.

Next, the inoculum is grown using the following air-temperature conditions:
time up to 12 h: temperature 38 ^o C, air consumption 4 m ³ / h,
time up to 24 hours: temperature 37 ^o C, air flow 5 m ³ / h.

 3.4. Basic fermentation.

In an industrial fermenter with a volume of 10 m ³ prepare 7 m ^{3 of the} nutrient medium of the following composition, g / DM ³ :
Glucose - 110
Magnesium Sulfate - 0.2
Manganese Sulfate - 0.05
Diammonium phosphate - 0.2
Monosubstituted potassium phosphate - 0.2
Artesian Water - Else
A swollen suspension of conidia is seeded on a sterile growth medium.

Next, they conduct the process using the following air-temperature conditions:
time up to 6 hours: temperature 37 ^o C, air flow 30 m ³ / h,
time up to 12 h: temperature 36 ^o C, air flow 40 m ³ / h,
time up to 24 hours: temperature 35 ^o C, air flow 50 m ³ / h.

 In the fermentation process, a 10 wt.% Suspension of calcium hydroxide maintain a pH of 3.5-4.5.

 At the end of sugar utilization, the process is stopped, the biomass is separated, the pH is adjusted with calcium hydroxide to pH 6.5 and 820 kg of calcium gluconate are obtained. The yield with respect to the spent sugar (785 kg) is 104.4% (theoretical yield of calcium gluconate monohydrate 113% with respect to glucose monohydrate).

 Example 4

 Obtaining glucono-δ-lactone.

 4.1. Obtaining seed in the form of conidia, as in example 1.

 4.2. Soaking conidia, as in example 2.

 4.3. Basic fermentation.

In a laboratory fermenter with a volume of 10 dm ³ , 7 dm ^{3 of a} nutrient medium of the following composition is prepared, g / dm ³ :
Glucose - 110
Magnesium Sulfate - 0.2
Manganese Sulfate - 0.05
Diammonium phosphate - 0.2
Monosubstituted potassium phosphate - 0.2
Artesian Water - Else
A swollen suspension of conidia is seeded on a sterile growth medium.

Next, they conduct the process using the following air-temperature conditions:
time up to 3 hours: temperature 37 ^o C, air flow 10 dm ³ / h,
time up to 6 hours: temperature 36 ^o C, air flow 20 dm ³ / h,
time up to 12 h: temperature 34 ^o C, air flow 25 dm ³ / h,
time up to 24 hours: temperature 34 ^o C, air flow 40 dm ³ / h.

 At the end of sugar utilization, the process is stopped, the biomass is separated, the pH is adjusted to 3.5 with a suspension of calcium hydroxide, and the precipitate is separated.

 Get 770 g of gluconic acid with the release to the spent sugar (790 g) of 97.5%.

Gluconic acid is isolated in the form of glucono-5-lactone after evaporation to 60 wt.% And crystallization at 40 ^o C.

 Taking into account losses during crystallization, the yield of glucono-δ-lactone with respect to sugar is 65%.

 Feasibility study of the strain VKPM F-790.

 The new VKPM strain F-790 expands the range of biological products synthesized by Aspergillus niger molds under industrial conditions.

Like the well-known strain L-4, it provides high technical and economic indicators in the production of citric acid (its salts). This valuable quality is combined with the ability with a higher sugar yield (by 17-22%) and productivity (by 20-30 kg / m ³ • day) in comparison with the known strain to produce gluconic acid and its derivatives.

 Gluconic acid biosynthesis with a new strain VKPM F-790 is a highly efficient process that has the character of biotransformation.

 Practical glucose losses are associated with its consumption for biomass accumulation and respiration. The high specificity of the reaction initiated by the VKPM strain F-790 leads to a decrease in the impurities of by-products compared to L-4 from about 15% to 1.5%, determined in the sum of the reaction products.

 The marked high resistance of the VKPM F-790 strain to acidic media in comparison with L-4 makes it possible to simplify the production of gluconic acid or gluconolactones.

 Due to the necessity of conducting the process of biosynthesis of gluconic acid derivatives at pH from 5.5 to 7, it is not possible to obtain gluconic acid or gluconolactones directly during biosynthesis using other gluconate producing strains. Usually [4] calcium gluconate is obtained, the acid decomposition of which produces gluconic acid. Strain VKPM F-790 allows biosynthesis at a pH of about 3.5 and obtain gluconic acid in the biosynthesis process.

 Thus, the VKPM strain F-790 allows you to rebuild industries that use Aspergillus niger mushroom for biosynthesis, focusing on a single product in flexible modern plants that are freely guided by requests and market conditions.

 Information materials.

 1. SU1811697 A3, C 12 N 1/14.

 2. SU 975799 C, C 12 N 15/08 (prototype).

 3. RU 2132878 C1, C 12 P 7/40.

 4. Industrial microbiology. / Ed. N.S. Egorova. - M .; p. 495 and 496, 498 and 499, 513 and 514.

Claims (1)

 The strain of the fungus Aspergillus niger VKPM F-790 is a producer of gluconic and citric acids.

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