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The EXAMPLE METHOD OF PURIFYING SUCCINIC ACID FROM FERMENTATION LIQUID
Example 1
Succinic acid was purified from a fermentation broth in which succinic acid(cas:110-15-6) diammonium salt was accumulated.
Preparation of H-type strongly acidic cation-exchange resin
H-type cation-exchange resin was prepared as follows.
1.6 L of Na-type strongly acidic cation-exchange resin SK1BL (Mitsubishi Chemical Corporation) was filled in a column. 5 L of 1 mol/L hydrochloric acid was passed through the column at a flow rate of 1.6 L/hr to convert the resin to H-type. Subsequently, 20 L of pure water was passed through the column to wash the resin, and the resin was used for the following experiments.
Production of succinic acid(cas:110-15-6) by fermentation
Succinic acid(cas:110-15-6) was produced by fermentation according to the following method.
400 mL of a medium which contains 100 g of glucose, 0.5 g of magnesium sulfate heptahydrate, 0.65 g of orthophosphoric acid, 14.3 mL of soybean protein hydrolysis solution (total nitrogen content: 35 g/L), 1.0 g of ammonium sulfate, 20 mg of ferrous sulfate heptahydrate, 20 mg of manganese sulfate hydrate, 1 mg of D-biotin, 1 mg of thiamin hydrochloride and 0.05 mL of anti-foam (GD-113, NOF Corporation) per 1 L was prepared, adjusted to pH 6.5 with 1 N KOH, poured into a 1-L jar fermenter and sterilized by heating at 120°C for 20 minutes. After cooling the medium, the Brevibacterium flavum MJ-233-AB-41 strain transformed with the plasmid pPC-PYC2 was inoculated in the medium, and the medium was maintained at 30°C. The culture was performed for 20 hours with aeration of 300 ml per minute and stirring at 700 rpm, while pH was adjusted to 7.6 with ammonia gas. 100 ml of the obtained culture solution was used for the following succinic acid(cas:110-15-6) fermentation.
79 ml of a succharide solution which contains 520 g of glucose and 2.6 g of magnesium sulfate heptahydrate per 1 L was prepared and sterilized by heating at 120°C for 20 minutes. 221 ml of a medium which contains 1.21 g of orthophosphoric acid, 5.39 mL of soybean protein hydrolysis solution (total nitrogen content: 35 g/L), 1.86 g of ammonium sulfate, 37.14 mg of ferrous sulfate heptahydrate, 37.14 mg of manganese sulfate hydrate, 1.86 mg of D-biotin, 1.86 mg of thiamin hydrochloride and 0.09 mL of anti-foam (GD-113) per 1 L was prepared, adjusted to pH 6.5 with 1 N KOH and then sterilized by heating at 120°C for 20 minutes. The sterilized saccharide solution and the sterilized medium were put into a 1-L jar fermenter, cooled, then added with 100 mL of the aforementioned culture broth to make the total volume 400 mL, and then maintained at 30°C. Succinic acid(cas:110-15-6) fermentation was performed for 24 hours with aeration of 20 ml per minute and stirring at 400 rpm, while the medium was adjusted to pH 7.6 with ammonia gas.
The above-described succinic acid(cas:110-15-6) fermentation was performed 9 times to obtain 3.6 L of culture broth having a succinic acid concentration of 25 g/L.
Purification of a crystal of succinic acid(cas:110-15-6) from fermentation broth in which salt of succinic acid(cas:110-15-6) was accumulated
The obtained culture broth was sterilized by heating at 120°C for 20 minutes and then centrifuged at 5000 x g for 20 minutes to obtain 3.5 L of supernatant.
The obtained supernatant was passed through the aforementioned H-type cation-exchange resin to obtain 2.2 L of a through-flow liquid. The obtained through-flow liquid was concentrated by using a rotary evaporator until the concentration of succinic acid(cas:110-15-6) became 19.2% to precipitate a crystal of succinic acid and obtain a succinic acid slurry. The obtained succinic acid slurry was cooled to 10°C to precipitate the crystal of succinic acid. The crystal and the mother liquor were separated. The obtained crystal of succinic acid was resuspended in a saturated aqueous solution of succinic acid having a volume of 15 times the weight of the crystal to wash the crystal, and then the crystal was separated.
Comparative Example 1
The fermentation broth in which succinic acid(cas:110-15-6) was accumulated in the same manner as in Example 1 was sterilized (120°C, 20 minutes), and the cells were removed (5000 x g, 20 minutes). This fermentation broth was concentrated by using a rotary evaporator so that succinic acid concentration became 234 g/L. The obtained concentrated liquid was adjusted to pH 2.2 with addition of sulfuric acid and cooled to 10 °C to precipitate crystal of succinic acid. Then, the crystal was separated in the same manner as in Example 1, and washed by reslurrying with a saturated aqueous succinic acid solution to obtain a washed crystal.
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