RU2458118C1 - Clostridium acetobutylicum strain - n-butanol producer - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: Clostridium acetobutylicum strain, the Russian National Collection of Industrial Microorganisms B-10289 is produced by the exposure to UV radiation of a bacterial suspension of the Clostridium acetobutylicum strain, No. 6 that is followed by the anaerobic steady-state selection. The n-butanol yield makes 8.3-14 g/l.

EFFECT: in the various fermentation environments, the strain is characterised by a higher level of n-butanol biosynthesis.

2 tbl, 6 ex

Description

The invention relates to the microbiological industry, in particular to the production of a strain producing n-butanol, used as an organic solvent, biofuel and the basis for the synthesis of many industrially valuable organic compounds.

Known strain VKPM B-4786, which on a substrate of 6% rye flour synthesizes up to 7.9 g / l n-butanol [1].

The closest analogue of the claimed strain is a strain of Clostridium acetobutylicum No. 6, which on a nutrient substrate from 6% rye flour synthesizes up to 8.3 g / l of n-butanol [1].

The objective of the invention is to obtain a strain of bacteria Clostridium acetobutylicum with an increased level of biosynthesis of n-butanol.

The problem was solved by obtaining a strain of Clostridium acetobutylicum CB 6-1, deposited in the All-Russian collection of industrial microorganisms (VKPM) as Clostridium acetobutylicum VKPM B-10289.

The inventive strain is obtained by irradiation with ultraviolet suspension of bacteria of the strain Clostridium acetobutylicum No. 6 and subsequent selection under anaerobic stationary conditions and has the following characteristics.

Cultural and morphological features

After 4 days of growth under anaerobic conditions on an agarized SOL medium (wt.%): KH ₂ PO ₄ - 0.07; K ₂ HPO ₄ - 0.07; MgSO ₄ · 7H ₂ O — 0.01; MnSO ₄ · 7H ₂ O — 0.002; FeSO ₄ · 7H ₂ O — 0.0015; NaCl - 0.001; ammonium acetate - 0.3; yeast extract - 0.1; peptone - 0.1; cysteine - 0.05; starch - 0.1; glucose 1; vitamin solution - 1 ml / l; resazurin - 1 mg / l; water - the rest; the colonies have a white center and a halo with smooth edges. Stroke on anaerobic agar: the surface is flat, the edges are flat.

Cells in the form of single elongated sticks.

Physiological signs

It ferments sugars: hexoses (glucose, galactose, mannose, fructose), pentose (xylose, arabinose), disaccharide (lactose, sucrose, maltose), oligosaccharide, starch, etc .; Peptone and ammonium are used as a source of nitrogen.

The strain Clostridium acetobutylicum VKPM B-10289 synthesizes n-butanol in an amount of up to 14 g / l on substrates containing rye flour. Resistant to up to 20 wt.% Culture liquids in the nutrient substrate after fermentation of strains of Clostridium tyrobutyricum VKPM B-10406 or Clostridium butyricum VKPMV-9619.

Storage conditions

The strain Clostridium acetobutylicum VKPM B-10289 is stored in laboratory No. 12 of the Federal State Unitary Enterprise GosNIIgenetika (address: 1 Dorozhny proezd, 1, 117545 Moscow) in a spore state on an environment of the following composition (wt.%): Peeled rye flour - 3 ; water - the rest, or on an MSS medium (wt.%): KH ₂ PO ₄ - 0.1; K ₂ HPO ₄ - 0.08; MgSO ₄ · 7H ₂ O - 0.1; FeSO ₄ · 7H ₂ O — 0.005; ammonium acetate - 0.3; yeast extract - 0.5; cysteine-HCl - 0.05; yeast extract - 0.5; para-aminobenzoic acid - 0.001; glucose - 2; water is the rest.

Reseeding is carried out six months later on one of the nutrient substrates mentioned above, thermostat at t = 37 ° C for 2-3 days. The resulting bacterial suspension is stored at t = 4-6 ° C.

In the All-Russian collection of industrial microorganisms, the strain Clostridium acetobutylicum VKPM B-10289 is stored in a lyophilized form.

Example 1. The biosynthesis of n-butanol of the claimed strain in a 6% aqueous suspension of rye flour

Fermentation is carried out in laboratory, static, strictly anaerobic conditions in bottles with a total volume of 120 cm ³ and a working volume of 50 cm ³ , the dose of seed 5% vol.

Fermentation conditions: pH 4.0-6.3, temperature 37 ° C, 48 hours. Characteristic of the nutrient substrate used: 6% aqueous suspension of rye flour with a starch content of 39 g / l.

As a control, the parent strain of Clostridium acetobutylicum No. 6, which is also the closest analogue, is used. The fermentation time is 72 hours.

The determination of the amount of n-butanol and other components of the culture fluid in all examples is carried out by gas chromatographic analysis.

From the results shown in table 1, it follows that on the substrate, which is a 6% aqueous suspension of rye flour, the claimed strain exceeds the closest analogue in terms of the level of biosynthesis of n-butanol (9.1 g / l instead (8.3 g / l )), as well as a number of other indicators given in the table.

Example 2. The biosynthesis of n-butanol of the inventive strain in a 6% aqueous suspension of rye flour with molasses in the amount of 0.5% sucrose

Fermentation conditions and control, as in example 1, the fermentation time is 48 hours for the inventive strain and 48 hours for control.

Characteristics of the nutrient substrate used: 6% aqueous suspension of rye flour with molasses in the amount of 0.5% sucrose (conditional starch content of 43.75 g / l).

From the results shown in table 1, it follows that on a substrate, which is a 6% aqueous suspension of rye flour with molasses in the amount of 0.5% in sucrose, the claimed strain exceeds the closest analogue in terms of the level of n-butanol biosynthesis (8.6 g / l instead of 8.3 g / l), as well as a number of other indicators given in the table.

Example 3. The biosynthesis of n-butanol of the claimed strain in a 6% aqueous suspension of rye flour with the addition of molasses in the amount of 1.35% RVI

Fermentation conditions and control, as in example 1, but the fermentation time for the claimed strain of 72 hours, and for control - 96 hours

Characteristics of the nutrient substrate used: 6% suspension of rye flour with molasses in the amount of 1.35% for reducing substances after inversion - RVI (content of conventional starch 51.8 g / l).

From the results shown in table 1, it follows that on a substrate, which is a 6% aqueous suspension of rye flour with molasses in the amount of 1.35% RVI, the claimed strain exceeds the closest analogue both in terms of the level of biosynthesis of n-butanol (14.0 g / l instead of 9.1 g / l), as well as a number of other indicators given in the table.

Example 4. The biosynthesis of n-butanol of the claimed strain in a 7% aqueous suspension of rye flour with the addition of molasses in the amount of 1.35% RVI

Fermentation conditions and control, as in example 1, but for control time 54 hours

Characteristics of the nutrient substrate used: 7% suspension of rye flour with molasses in the amount of 1.35% by RVI (content of conventional starch 58.3 g / l).

From the results shown in table 1, it follows that on a substrate, which is a 7% aqueous suspension of rye flour with molasses in the amount of 1.35% RVI, the claimed strain exceeds the closest analogue in terms of the level of n-butanol biosynthesis (13.5 g / l instead of 12.3 g / l), as well as a number of other indicators given in the table.

Example 5. The biosynthesis of n-butanol of the inventive strain on rye flour with the addition of culture fluid of a strain of Clostridium tyrobutyricum VKPM B-10406

Prepare the seed of the strain Clostridium tyrobutyricum VKPM-10406. To do this, use the nutrient substrate SOL of the following composition (wt.%): KH ₂ PO ₄ - 0.07; K ₂ HPO ₄ - 0.07; MgSO ₄ · 7H ₂ O — 0.01; MnSO ₄ · 7H ₂ O — 0.002; FeSO ₄ · 7H ₂ O — 0.0015; NaCl - 0.001; ammonium acetate - 0.3; yeast extract - 0.1; peptone - 0.1; cysteine - 0.05; vitamin solution - 1 ml / l; water is the rest. The content of monosaccharides is 2%. As sources of monosaccharides, an enzymatic hydrolyzate of plant materials (corn cob, etc.) with inverted molasses additives in an amount of 1% sucrose is used (molasses is used as a source of biotin). The nutrient substrate is sterilized at t = 125 ° C for 1 hour. Cultivation is carried out for 7 days.

The resulting seed in an amount of 10 vol.% Seeded in a bioreactor containing a nutrient substrate above the specified composition with a concentration of PB = 2 wt.% And carry out the process of fermentation for 10 days.

When preparing the seed and carrying out the fermentation of oleic acid, the following technological regime is used: t = 37 ° C, pH 5.2-6.5. The pH of the medium is preferably controlled with a 25% solution of ammonia water.

The grown bacterial biomass is separated by centrifugation or separation (5-10 rpm). The centrate is used in the preparation of a nutrient substrate for the process of acetone-butanol fermentation in an amount of 20 wt.%.

For acetone-butanol fermentation, the strain Clostridium acetobutylicum VKPM B-10289 (CB 6-1) is used. To obtain seed, the culture is seeded in an amount of 5 vol.% In relation to the volume of the substrate. The substrate is sterilized at a temperature of 135 ° C for 1 hour. Fermentation is carried out for 48 hours.

The obtained seed in an amount of 3 vol.% Seeded in a bioreactor with a sterile nutrient substrate. The composition of the substrate (wt.%): Rye flour - 6, molasses - 0.5 for sucrose, the culture fluid after fermentation with Clostridium tyrobutiricum strain VKPM-10406 - 20, water - the rest (conditional starch in the substrate 4.375 wt.%). Fermentation time is 48 hours.

When preparing the seed and carrying out acetone-butanol fermentation, the following technological regime is used: t = 37 ° C, pH 4.0-6.3. The pH of the medium is preferably controlled with a 25% solution of ammonia water.

From the results shown in table 2, it follows that under the proposed conditions the claimed strain exceeds the closest analogue in the level of biosynthesis of n-butanol (8.9 g / l instead of 4.4 g / l), as well as a number of other indicators given in table 2 .

Example 6. The biosynthesis of n-butanol of the inventive strain on rye flour with the addition of culture fluid of a strain of Clostridium butyricum VKPM B-9619

The technological scheme, substrates and fermentation conditions are similar to those described in Example 5. To carry out the process of acetone-butanol fermentation, a nutrient substrate is used with the addition of culture fluid of the Clostridium butiricum VKPM B-9619 strain in an amount of 20 wt.%. For acetone-butanol fermentation, the strain Clostridium acetobutylicum VKPM B-10289 is used.

From the results shown in table 2, it follows that under the proposed conditions the claimed strain exceeds the closest analogue in the level of biosynthesis of n-butanol (9.9 g / l instead of 0.15 g / l), as well as a number of other indicators given in table 2 .

Thus, the claimed strain of Clostridium acetobutylicum VKPM B-10289 in all the proposed fermentation conditions exceeds the closest analogue in the level of biosynthesis of n-butanol, and its fermentation time is either shorter or corresponds to that of the closest analogue.

Information sources

1. Berezina O. V., Sineokiy S. P., Velikodvorskaya G. A. et al. Extracellular glycosyl hydrolase activity of clostridia forming acetone, butanol and ethanol // Applied Biochemistry and Microbiology. - 2008. - T.44. - No. 1.

Claims (1)

The bacterial strain Clostridium acetobutylicum CB 6-1, VKPM B-10289 is a producer of n-butanol.