The strain of the fungus aspergillus niger vkpm f - 790 producing gluconic and citric acids

 

(57) Abstract:

The invention relates to the microbiological industry and relates to a new strain of the fungus Aspergillus niger VKPM F-790. Depending on the creation of conditions strain produces citric acid or its salts and gluconic acid (in the form of its salts or gluconolactone). Strain VKPM F-790 allows you to rebuild the production, use for biosynthesis of the fungus Aspergillus niger, get monoproduct a few products that are economically more profitable. The new strain is more stable in acidic environments, which allows to simplify the production of gluconic acid. table 1.

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

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

In the process of biosynthesis of citric acid these strains produces about 10% gluconic acid to cya gluconic acid or its derivatives such indicators biosynthesis of no interest.

When providing special, favorable for the formation of gluconic acid or its derivatives [3], the output of these products increases up to 75-85% of the introduced sugar. As byproducts of citric and oxalic acid, complicating the selection of the target product and lost forever.

The present invention is to obtain a new strain of the fungus Aspergillus niger with high activity and productivity as in the biosynthesis of citric acid (and its salts), and in the process of obtaining gluconic acid, its salts or gluconolactone) in an industrial environment.

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

For five years in the industrial production of citric acid made the selection, screening and isolation of samples of culture L-4 exposed in vivo production of mutations, which was accompanied by increased accumulation of acid, increased resistance to bacterial and yeast infection.

Special attention is drawn samples of culture, providing along with high technical and economic indicators in Bisevo acid and its derivatives.

About the value of the target products of biosynthesis, the production of which is possible on the basis of application of the new strain, can be judged from the information presented in the work [4].

The resulting breeding strain of the fungus Aspergillus niger has a collection number VKPM F-790.

Cultural morphological traits studied in pachystachya crops grown in synthetic medium of čapek or wort-agar nutrient medium.

Cultural characteristics on the environment of čapek.

The colony has a diameter 782 mm, color beige, in the centre of yellowish, slightly raised above the substrate. Conidial heads in the center of the colony large, are tight, form the hill to the edge of their size decreases. Size asporogenic zone is about 6 mm Reverse side of the colony folded, white.

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

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

Cultural characteristics on wort-agar nutrient medium.

The colony has a diameter 951 mm Colour beige. Aerial mycelium is well developed. 5 mm The reverse side of the colony folded, white.

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

Morphological features identified in pachystachya crops grown in the environment of čapek.

Conidial heads round shape, their diameter 11061 μm, swelling of canadianese spherical shape with a diameter of 314 mm. Sterigma double layer: sterigma first order length 155 μm, sterigma second order length 7,52,5 μm, the surface is covered with protuberances. Conidia chained flattened sides, devoid of protuberances. The average size of conidia 3,30,3 μm. Conidiophores, hyaline, length of conidiophores 867561 μm, the diameter 10,53,5 microns.

Comparison with known strain L-4 shows that the strain VKPM F-790 is large conidial heads, less the amount of swelling of the conidiophores, the length of sterigma first order, the size of the conidia and the diameter of canadianese (see table).

Among the biochemical properties of the strain VKPM F-790 deserves mention for its ability to grow and biosynthesis in more acidic environments than the original strain. In-depth interviews kultiviert known strain L-4 (optimum pH at similar application of 5.5 to 6.0). Perhaps this is a sign of strain VKPM F-790 provides greater stability compared to the L-4 to bacterial and yeast infection.

A new strain VKPM F-790 as well as well-known L-4, - aerobe, prototroph. Capable of fermentation from 20 hours to 12 days. Well kept and reproduces its properties in the form of dry conidia (residual moisture 5-10 Rel.%).

The activity and productivity of a new strain VKPM F-790 appreciate in industrial (biosynthesis of citric acid and calcium gluconate) and laboratory (biosynthesis salts of citric acid and gluconolactone) conditions.

Under activity understand the yield of the target product obtained in relation to the Sahara for a certain time of fermentation. About the efficiency of a process is judged by the volume of product biosynthesis per unit volume of reactor per day.

As can be seen from the table, with close values of the activity and productivity of new and known strains in the biosynthesis of citric acid strain VKPM F-790 at 17-22% active and 25-30 kg/m3day more productive L-4 during the production of gluconic acid or its derivatives. These figures are accompanied by lower concentrations of by-products of biosynthesis (see table).

The sources of carbon.

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

The inventive explain some specific examples of the use of strain VKPM F-790.

Example 1.

Obtaining citric acid.

1.1. Getting the seed in the form of conidia.

Conidia grown in chambers thermostats with adjustable temperature and humidity on the cuvettes filled with a layer of 1.5 cm nutrient medium composition, g/DM3:

Beer wort - 20

Urea - 1

Sodium chloride - 20

Agar - 20

Artesian water - the Rest

pH to 5.8 before sterilization.

The environment is seeded with a culture of the strain F-790 and incubated for 7 days under conditions of forced aeration with sterile air, maintaining the following mode of cultivation:

1-4 day: humidity is reduced from 80 to 60 Rel.%;

day 5-7: humidity is reduced from 60 to 40 Rel.%.

On the 8th day the temperature was lowered to 18oWith and collect conidia vacuum device. Dried conidia when the 32oWith up to a residual moisture content of 7 Rel.%.

1.2. terasawa 50 g of molasses.

Maintain suspension of conidia on the rocking chair with the number of swings 160 min for 7 h at 34oC.

1.3. Growing seed - inoculum.

In industrial seed fermenter - inoculator volume of 10 m3prepare a nutrient medium of the following composition, g/DM3:

Molasses - 50

Ferrocyanide of potassium - 0,25

The ammonium oxalate - 2

Zinc sulfate - 0,005

Magnesium sulfate - 0,25

Potassium phosphate one-deputizing - 0,16

Artesian water - the Rest

Sow on the prepared nutrient medium swollen suspension of conidia.

Next seed, the AMF inoculum, grown using the following air-temperature range:

time up to 12 h: temperature 38oC, air flow 200 m3/h;

time up to 24 hours: temperature 37oC, air flow 300 m3/PM

1.4. The main fermentation.

In an industrial fermenter with a volume of 100 m3cook 60 m3the nutrient medium of the following composition, g/DM3:

Molasses - 60

Ferrocyanide of potassium - 0,25

The ammonium oxalate - 2

Zinc sulfate - 0,005

Magnesium sulfate - 0,25

Potassium phosphate one-deputizing - 0,16

Artesian water - the Rest

pH 5.5.


day 1: air flow 1200 m3/h, the temperature of the 37oC;

day 2: air flow 1500 m3/h, the temperature of the 36oC;

day 3: air flow 1800 m3/h, the temperature of the 36oC;

day 4: air flow rate of 2000 m3/h, the temperature of the 34oC;

day 5: air flow 2200 m3/h, the temperature of the 34oC.

Since 3 days, produce dolive 0.25 m3the nutrient medium of the following composition, g/DM3:

Molasses - 500

Ferrocyanide of potassium - 0,5

The ammonium oxalate - 3

Artesian water - the Rest

After separation of the biomass receive 68 m3solution with a total acidity of 10 wt.% and the concentration of citric acid 9 wt.% (6120 kg). Access to the spent sugar (8100 kg) of 75.6%.

Example 2.

Getting citrate.

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

2.2. Soaking conidia.

0.1 g of conidia soaked 700 cm3water-molasses medium containing 40 g of molasses.

Maintain the suspension on the rocking chair with the number of swings 160 min for 7 h at 34oC.

2.3. The main fermentation.

In laboratory fermenter with a volume of 10 DM3cook 7 d of magnesium Sulfate 0,25

DAP - 0,4

Potassium phosphate one-deputizing - 0,16

Urea - 2

Artesian water - the Rest

Seeded on sterile nutrient medium swollen suspension of conidia.

Next, lead the process, using the following air-temperature range:

day 1: temperature 38oC, air flow 100 DM3/h,

day 2: temperature 36oC, air flow 150 DM3/h,

day 3-5: temperature 34oC, air flow 220 DM3/PM

A solution of 10 wt.% sodium hydroxide, since 3 days, maintain a pH of about 3.5.

Upon completion of the disposal of sugar the stop process, the biomass is separated, the pH of the solution was adjusted to 6.5. Get 810 g of sodium citrate. Output in relation to the used sugar (1070 g) to 75.7%.

Example 3.

Receiving calcium gluconate.

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

3.2. Growing seed - inoculum.

In industrial seed fermenter - inoculator volume of 1 m3prepare 600 DM3the nutrient medium of the following composition, g/DM3:

Glucose - 25

Magnesium sulfate - 0,2

Manganese sulfate - 0,05

Diamon swollen suspension of conidia on the prepared sterile nutrient medium.

Further, the AMF inoculum is grown, using the following air-temperature range:

time up to 12 h: temperature 38oC, air flow rate of 4 m3/h,

time up to 24 hours: temperature 37oC, air flow 5 m3/PM

3.4. The main fermentation.

In an industrial fermenter with a volume of 10 m3cook 7 m3the nutrient medium of the following composition, g/DM3:

Glucose - 110

Magnesium sulfate - 0,2

Manganese sulfate - 0,05

DAP - 0,2

Potassium phosphate one-deputizing - 0,2

Artesian water - the Rest

Seeded on sterile nutrient medium swollen suspension of conidia.

Next, lead the process, using the following air-temperature range:

time up to 6 h: temperature 37oC, air flow 30 m3/h,

time up to 12 h: temperature 36oC, air flow rate of 40 m3/h,

time up to 24 hours: temperature 35oC, air flow 50 m3/PM

In the fermentation process 10 wt.% suspension of calcium hydroxide maintain pH 3,5-4,5.

Upon completion of the disposal of sugar the stop process, the biomass is separated, adjusted pH of calcium hydroxide to pH 6.5 and receive 820 kg of calcium gluconate. The output p is s to the glucose monohydrate).

Example 4.

Getting glucono-lactone.

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

4.2. Soaking conidia as in example 2.

4.3. The main fermentation.

In laboratory fermenter with a volume of 10 DM3cook 7 DM3the nutrient medium of the following composition, g/DM3:

Glucose - 110

Magnesium sulfate - 0,2

Manganese sulfate - 0,05

DAP - 0,2

Potassium phosphate one-deputizing - 0,2

Artesian water - the Rest

Seeded on sterile nutrient medium swollen suspension of conidia.

Next, lead the process, using the following air-temperature range:

time up to 3 h: temperature 37oC, air consumption 10 DM3/h,

time up to 6 h: temperature 36oC, air flow 20 DM3/h,

time up to 12 h: temperature 34oC, air flow rate of 25 DM3/h,

time up to 24 hours: temperature 34oC, air flow 40 DM3/PM

Upon completion of the disposal of sugar the stop process, the biomass is separated, adjusted pH to 3.5 suspension of calcium hydroxide, the precipitate was separated.

Get 770 g of gluconic acid with access to the spent sugar (790 g) 97 the 40oC.

Taking into account losses during crystallization output glucono-lactone in relation to sugar is 65%.

Technical and economic efficiency of the strain VKPM F-790.

A new strain VKPM F-790 extends the range of bioproducts, synthesized mold fungi of the species Aspergillus niger in an industrial environment.

Like the well-known strain L-4 it provides high technical and economic indicators in the production of citric acid (and its salts). This is a valuable quality combined with an ability with a high yield of sugar (17-22%) and productivity (20-30 kg/m3day) in comparison with the known strain to produce gluconic acid and its derivatives.

Biosynthesis of gluconic acid by a new strain VKPM F-790 is a highly effective process, having the character of biotransformation.

Practical loss of glucose associated with its consumption on the accumulation of biomass and respiration. The high specificity of the reaction, initiated by the strain VKPM F-790, reduces impurities by-products compared to L-4 from about 15% to 1.5%, a certain amount of reaction products.

Noted most resistant strain VKPM F-790 to the acidic environment in comparison with L-4 allows the ECCA biosynthesis of derivatives of gluconic acid at a pH of from 5.5 to 7 using other strains-producers gluconate cannot get gluconic acid or gluconolactone during biosynthesis. Usually [4] get calcium gluconate, acid decomposition which receive gluconic acid. Strain VKPM F-790 allows biosynthesis at a pH of about 3.5 and get gluconic acid biosynthesis.

Thus, the strain VKPM F-790 allows you to rebuild the production, use for biosynthesis of the fungus Aspergillus niger, with a focus on monoproduct flexible in modern production, being oriented on demand and market conditions.

Informational materials.

1. SU1811697 A3, C 12 N 1/14.

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

3. EN 2132878 C1, C 12 P 7/40.

4. Industrial Microbiology. / Ed. by N. With. Egorova. - M.; S. 495 and 496, 498 and 499, 513 and 514.

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

 

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FIELD: biotechnology, microbiology, organic chemistry.

SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.

EFFECT: improved preparing method.

1 tbl, 6 ex

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