RU2455350C1 - Clostridium acetobutylicum STRAIN - n-BUTANOL PRODUCER - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Clostridium acetobutylicum strain, the Russian National Collection of Industrial Microorganisms B-10290 is produced by the exposure to UV radiation of a bacterial suspension of the Clostridium acetobutylicum strain, the Russian National Collection of Industrial Microorganisms B-4786, that is followed by the anaerobic steady-state selection. The n-butanol yield makes up to 20 g/l on a medium consisting of a rye flour suspension with added molasses and maltose.

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

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Description

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

A known strain of Clostridium acetobutylicum VKPM B-4786, which synthesizes 7.9 g / l of n-butanol on a nutrient substrate from a 6% aqueous suspension of rye flour [1].

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

As a control, we will use the Clostridium acetobutylicum VKPM B-10289 strain, which synthesizes n-butanol in an amount of up to 14 g / l on a 6% aqueous suspension of rye flour with the addition of 1.35% RVI molasses (RF Patent Application 2011105882).

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-2, deposited in the All-Russian collection of industrial microorganisms (VKPM) as Clostridium acetobutylicum VKPM B-10290.

The inventive strain is obtained by irradiation with ultraviolet suspension of bacteria of the strain Clostridium acetobutylicum VKPM B-4786, followed by selection under anaerobic stationary conditions and has the following characteristics.

Cultural and morphological properties

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 are flat, the center of the colony is less pronounced, looks like an entire colony without a halo, the edges are uneven. Stroke: the surface is flat, the edges are flat.

Cells in the form of single elongated sticks.

Physiological properties

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-10290 synthesizes n-butanol in an amount of up to 20 g / l on substrates consisting of a suspension of rye flour with the addition of molasses and maltose.

Resistant to up to 30 wt.% Culture liquids in the nutrient substrate after butyric fermentation of strains of Clostridium tyrobutyricum VKPM B-10406 or Clostridium butyricum VKPM B-9619.

Storage conditions

The strain Clostridium acetobutylicum VKPM B-10290 is stored in laboratory No. 12 of the Federal State Unitary Enterprise GosNIIgenetika (address: 1 Dorozhny proezd 1, Moscow, 117545) in a spore state on a medium of the following composition (wt.%): Peeled rye flour - 3; water - the rest or MSS medium (wt.%): KH ₂ PO ₄ - 0.1; To 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 other water.

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-10290 is stored in a lyophilized form.

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.

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

The fermentation of the inventive strain is carried out in laboratory, static, strictly anaerobic conditions in vials with a total volume of 120 cm ³ and a working volume of 50 cm ³ , the seed dose of 4 vol.% At pH 4.0-6.3, temperature 37 ° C, for 96 hours

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% sucrose, the claimed strain exceeds the closest analogue, parent and control strains in terms of the level of biosynthesis of n- butanol (9.4 instead of 8.3, 8.4 and 8.5 g / l, respectively), 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 7% aqueous suspension of rye flour

Fermentation conditions, as in example 1, but the fermentation time for the claimed strain is 54 hours, for control and the closest analogue - 96 hours, for the parent 120 hours

Characteristics of the nutrient substrate used: 7% aqueous suspension of rye flour (starch content 45.5 g / l).

From the results shown in table 1, it follows that on the substrate, which is a 7% aqueous suspension of rye flour, the claimed strain is superior to the closest analogue, parent and control strains in terms of the level of n-butanol biosynthesis (12 g / l instead of 8.6, 9.0 and 8.8 g / l, respectively), as well as a number of other indicators given in the table with a shorter fermentation time.

Example 3. The biosynthesis of n-butanol of the claimed strain in a 7% aqueous suspension of rye flour with the addition of molasses and maltose

Fermentation conditions, as in example 1, but the fermentation time for the inventive strain is 96 hours

Characterization of the nutrient substrate used: 7% suspension of rye flour with molasses in the amount of 0.5% sucrose and 1% maltose (conditional starch content 59.75 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 0.5% sucrose and 1% maltose, the claimed strain has high rates both in terms of the level of n-butanol biosynthesis (20 g / l), as well as a number of other indicators given in the table.

The control strain and the closest analogue strain do not ferment such a substrate.

Example 4. The biosynthesis of n-butanol of the inventive strain on a substrate containing potatoes and glucose

Fermentation conditions, as in example 1, but within 48 hours. Residual glucose - 0.74 wt.%.

The characteristic of the used nutrient substrate (wt.%): Potato - 25.0 g / l (75% humidity, 18% starch), ammonium acetate - 0.15; chalk - 0.2; cysteine - 0.05; glucose - 5.0 (∑56.3 g / l of conditional starch).

From the results shown in table 1, it follows that on a substrate containing potatoes and glucose, the claimed strain has high rates both in terms of the level of biosynthesis of n-butanol (12.2 g / l), and a number of other indicators shown in the table. The control strain and the closest analogue strain do not ferment such a substrate.

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 in an amount of 8 wt.%

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; To 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 - 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. The pH of the medium is preferably controlled with a 25% solution of ammonia water.

The grown bacterial biomass is separated by centrifugation (10 rpm).

The centrate is used in the preparation of a nutrient substrate for the process of acetone-butyl fermentation in an amount of 8 wt.%.

For acetone-butyl fermentation, the strain Clostridium acetobutylicum VKPM B-10290 (CB-2) is used. To obtain seed, the culture is sown in an amount of 4% vol. In relation to the volume of the substrate of the following composition: 6% suspension of rye flour, molasses in an amount of 0.5% sucrose (conditional starch in the substrate 43.75 wt.%) And cultivated in within 48 hours

The resulting seed in an amount of 4 vol.% Seeded in a bioreactor with a sterile nutrient substrate. The composition of the substrate: 6% suspension of rye flour, molasses in an amount of 0.5% sucrose (conditional starch in the substrate 43.75 wt.%), 8 wt.% Of the culture fluid after oily fermentation with a strain of Clostridium tyrobutiricum VKPM-10406.

The process of acetone-butyl fermentation is carried out for 48 hours

When preparing the seed and carrying out acetone-butyl 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 in the proposed conditions the inventive strain has high levels of synthesis of n-butanol (9.52 g / l), as well as a number of other indicators shown 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 tyrobutyricum VKPM B-10406 in an amount of 20 wt.% For 48 hours

Fermentation conditions, as in example 5, but with the addition of culture fluid (CL) of the strain Clostridium tyrobutyricum VKPM B-10406 in an amount of 20 wt.%.

The strain Clostridium tyrobutyricum No. 6, the closest analogue, was used as a control.

From the results shown in table 2, it follows that in the proposed conditions, the inventive strain exceeds the closest analogue in the level of synthesis of n-butanol (5.42 g / l instead of 4.4 g / l).

table 2 The content of QOL in the nutrient substrate, strain Time h The chemical composition of the spent culture fluid, g / l Yield from conditional starch,% butanol acetone ethanol acid butanol solvents oil vinegar Substrate - 6% aqueous suspension of rye flour with the addition of molasses and QoL Clostridium tyrobutyricum VKPM-10406 QOL 8 wt.% Example No. 5 Strain VKPM B-10290 (claimed) 48 9.52 4.75 1,04 0.67 1.39 21.8 35.0 QOL 20 wt.% Example No. 6 Strain VKPM B-10290 (claimed) 48 5.42 3.25 0.32 2.17 1,69 QOL 20 wt.% Example No. 6 Strain No. 6 (the closest analogue) 48 4.4 2.7 0.3 2.16 1.82 10.0 16.9 QOL 20 wt.% Example No. 7 Strain VKPM B-10290 (claimed) 96 11.02 5.77 0.69 1.66 2,37 25,2 40,0 QOL 30 wt.% Example 8 Strain VKPM B-10290 (claimed) 96 10,2 5.10 0.83 0.89 1.73 23.3 36.9 The substrate is a 6% aqueous suspension of rye flour with the addition of molasses and QoL Clostridium butyricum VKPM B-9619 in an amount of 20 wt.% Strain VKPM B-10290 (claimed) 48 10,4 5.7 0.7 0.8 1,6 23.8 38,4 Strain VKPM B-10289 (control) 48 9.90 5.19 0.72 1.39 1,67 22.6 36.1 Strain No. 6 (closest analogue) 48 0.15 0.04 0,03 0.95 0.83 0.3 0.5

Example 7. 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 in an amount of 20 wt.% For 96 hours

The fermentation conditions, as in example 6, but for 96 hours. From the results shown in table 2, it follows that in the proposed conditions the inventive strain has high levels of synthesis of n-butanol (11.02 g / l), and a number of other indicators shown in table 2.

Example 8. The biosynthesis of n-butanol of the claimed strain on rye flour with the addition of culture fluid of a strain of Clostridium tyrobutyricum VKPM B-10406 in an amount of 30 wt.%

Fermentation conditions, as in example 7, but with the addition of culture fluid of the strain Clostridium tyrobutyricum VKPM B-10406 in an amount of 30 wt.%.

From the results shown in table 2, it follows that in the proposed conditions, the inventive strain has high levels of synthesis of n-butanol (10.02 g / l), as well as a number of other indicators shown in table 2.

Example 9. 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-9619

The technological scheme and substrates are similar to the technological scheme and substrates described in example 5, but for the process of acetone-butyl fermentation, a nutrient substrate is used with the addition of culture fluid of the Clostridium butyricum VKPM B-9619 strain in an amount of 20 wt.%. For acetone-butyl fermentation, the strain Clostridium acetobutylicum VKPM B-10290 (CB-2) is used. Fermentation time 48 hours

From the results shown in table 2, it follows that in the proposed conditions the claimed strain exceeds the level of synthesis of n-butanol as the closest analogue and control (10.4 g / l instead of 0.15 g / l and 9.9 g / l, respectively), and also surpasses the closest analogue in a number of other indicators shown in table 2.

Thus, the claimed strain of Clostridium acetobutylicum VKPM B-10290 in all the proposed fermentation conditions exceeds the closest analogue in the level of biosynthesis of n-butanol.

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-2, VKPM B-10290 is a producer of n-butanol.