Succinic acid-producing strain and its screening method and uses
A technology for producing succinic acid and strains, which is applied in the field of bioengineering and can solve problems such as difficulty in cultivation
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Embodiment 1
[0042] Weigh 1.0 g of the strain-containing mixture obtained from the rumen of the cow, and add it to 50 ml of sterile water to prepare a rumen bacteria suspension. Take 1ml of rumen bacteria suspension into a 100ml serum bottle, the amount of enrichment medium in the serum bottle is 50ml, and the composition of the enrichment medium is: NaCl 1.0g / L, K 2 HPO 4 4.0g / L, MgCl 2 ·6H 2 O 0.05g / L, CaCl 2 ·H 2 O0.05g / L, (NH 4 ) 2 SO 4 0.2g / L, yeast extract 10g / L, peptone 5.0g / L, glucose 50.0g / L, monensin 0.2mg / L, disodium fumarate 20.0g / L, disodium succinate 60.0 g / L, magnesium carbonate 50g / L; after enrichment culture at 37°C for 18h, the culture sample was diluted 10 -6 , take a 0.1ml droplet spread on the flat plate containing the separation medium, and cultivate it in an anaerobic incubator. 12 20mg / L, lipoic acid 20mg / L, niacin 20mg / L, pantothenic acid 20mg / L, thiamine 20mg / L, vitamin B6 20mg / L, agar 20g / L; mixed gas N in the anaerobic incubator 2 :CO 2 :H 2 =8:1:...
Embodiment 2
[0050] Seed medium: yeast extract 5g / L, peptone 5g / L, NaCl 1g / L, NaHCO 3 10g / L, Na 2 HPO 4 9~15g / L,KH 2 PO 4 4~12g / L, add agar 20g / L when cultivating on a slant surface, and inject N 2 :CO 2 =4:1 mixed gas for 2 minutes, sterilized at 121°C for 20 minutes, and cooled.
[0051] Fermentation medium: glucose 100g / L, MnCl 2 0.05g / L, FeCl 2 0.05g / L, CaCl 2 0.05g / L, K 2 HPO 4 2g / L, MgCO 3 100g / L, vitamins added folic acid 20mg / L, biotin 20mg / L, vitamin B 12 20mg / L, lipoic acid 20mg / L, niacin 20mg / L, pantothenic acid 20mg / L, thiamine 20mg / L, vitamin B 6 20mg / L, yeast extract and peptone respectively use the following dosages:
[0052] (1) 10g / L yeast extract, 5g / L peptone;
[0053] (2) 15g / L yeast extract, 5g / L peptone;
[0054] (3) 10g / L yeast extract, 15g / L peptone;
[0055] The volume of 100ml serum bottle is 50ml, and N 2 :CO 2 =4:1 mixed gas for 2 minutes, sterilized at 121°C for 20 minutes, and cooled.
[0056] After streaking the strain with the pre...
Embodiment 3
[0061] Seed medium: yeast extract 5g / L, peptone 5g / L, NaCl 1g / L, NaHCO 3 10g / L, Na 2 HPO 4 9~15g / L,KH 2 PO 4 4~12g / L, (add agar 20g / L when cultivating on a slant), pass through N 2 :CO 2 = 4:1 mixed gas for 2 minutes, sterilized at 121°C for 20 minutes, and cooled.
[0062] Fermentation medium: glucose 100g / L, yeast extract 10-50g / L, peptone 10-50g / L, MnCl 2 0.05g / L, FeCl 2 0.05g / L, CaCl 2 0.05g / L, K 2 HPO 4 2.0g / L, MgCO 3 100g / L, vitamins added folic acid 5mg / L, biotin 5mg / L, vitamin B 125mg / L, lipoic acid 5mg / L, niacin 5mg / L, pantothenic acid 5mg / L, thiamine 5mg / L, vitamin B 6 5mg / L, pH7.0.
[0063] When cultivating in a 5L fermenter, the liquid volume of the medium is 3L, with 100g / L MgCO 3 As a pH buffer, the fermenter gas mixture (N 2 :CO 2 =4:1) The ventilation rate is 0.6L / min to maintain the anaerobic environment of the fermentation system. After streaking the strain with the preservation number CGMCC No.1716 on the seed slant medium, culture ...
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